Break-induced replication is enhanced by a phospho-activated RPA-binding module in Pol32

Domain: neuroscience
Published: 2026-04-09
Editorial outcome: major_revision

Abstract
Break-induced replication (BIR) facilitates single-ended DNA double-strand break (DSB) repair.
Upon homologous recombination-mediated strand invasion into a homologous repair template,
BIR is catalysed by a minimal replisome comprising PCNA, DNA polymerase \u03b4 (Pol \u03b4), and the
Pif1 helicase. Here, we identify an interaction between Pol \u03b4 and single-stranded DNA (ssDNA)-
binding protein RPA mediated by an RPA-binding module (RBM) within Pol \u03b4 subunit Pol32 and
RPA subunit Rfa1. Pol32 RBM phosphorylation at Thr256 and Thr257 increases its affinity for
Rfa1, while corresponding phospho-mimetic amino-acid substitutions promote BIR efficiency in
vivo. This suggests that Pol32 functions as a rheostat whose phosphorylation enhances Pol \u03b4\u2019s
affinity for RPA-bound BIR intermediates, thereby boosting BIR efficiency. Modelling indicates that
Pol32 phospho-RBM-Rfa1 interactions mirror the binding mode of RBMs in Pif1 and the FANCM-
family helicase and BIR antagonist Mph1. This implies a key role for RPA in the dynamic
orchestration of the enzymes mediating BIR.

Introduction
Homologous recombination mediates error-free DNA double-strand break (DSB) repair by
harnessing a template with DNA sequence homology across the break site to fill the gap by repair
synthesis. At single-ended DSBs, which can be generated by DNA replication fork breakage or
telomere erosion, a similar repair process known as break-induced replication (BIR) occurs.1-4
First, DNA end-resection exposes a 3'-single-stranded DNA (ssDNA) overhang at the break site.
Rad51 then forms a nucleoprotein filament on the ssDNA and mediates strand invasion at a repair
template with sequence homology. This displaces one of the DNA strands at the repair template
to form a displacement loop (D-loop). The invading strand serves as a primer for DNA synthesis,
which is mediated by a BIR replisome consisting of DNA polymerase \u03b4 (Pol \u03b4), the processivity
clamp PCNA, and the helicase Pif1. A double-edged sword, BIR can rescue chromosome
replication after replication fork breakage but is prone to mutations, ectopic recombination and
chromosome aberrations.5-8 This suggests that cells must exercise careful control over BIR, and
there is evidence that cells limit the extent of BIR-dependent DNA synthesis during the
recombinational repair of Cas9 nickase-induced replication fork collapse. This can be achieved
through active D-loop processing by structure-specific nucleases and the arrival of a converging
DNA replication fork.2,9 Alternatively, cells may suppress BIR to favour the generation of a two-
ended DSB and subsequent repair by error-free homologous recombination.10-12

In contrast to canonical replication, BIR exposes extensive amounts of ssDNA, both by
strand displacement at the D-loop and in the form of the spooled-out nascent strand trailing the
progressing D-loop. The nascent strand remains temporarily single-stranded before serving as
template for lagging-strand synthesis. In the cell, ssDNA is quickly covered by the trimeric ssDNA-
binding protein RPA consisting of Rfa1/RPA70 (yeast/human), Rfa2/RPA32, and Rfa3/RPA14.
The Rfa1/RPA70 N-terminal domain (NTD) harbours a basic-hydrophobic groove that serves as
a hub for various client proteins with small acidic-hydrophobic RPA-binding modules (RBMs).
RBMs have been identified in several DNA damage response factors and shown to facilitate DNA
metabolic processes during DNA replication, repair, and recombination.13-21 Recently, an RBM
was identified within helicase Pif1, which is required for D-loop progression during BIR.20,22
Whether there is direct crosstalk between PCNA-Pol \u03b4 and RPA during BIR is not known.

Saccharomyces cerevisiae Pol \u03b4 consists of the catalytic subunit, Pol3, and the accessory
subunits Pol31 and Pol32. Pol32 was found to be dispensable for cell viability in budding yeast,
but is required for efficient BIR.23 Thus, pol32\u2206 cells initiate DNA synthesis but cannot sustain BIR
over long distances, so Pol32 can be described as a processivity factor for Pol \u03b4 in the context of
a D-loop.24 A cryo-EM structure of the Pol \u03b4 holoenzyme shows that the Pol32 NTD spanning
amino acid residues (aa) 1-115 folds into a globular domain that closely associates with Pol31,
which in turn binds the catalytic subunit Pol3.25 Loss of Pol32 compromises Pol \u03b4 function and
results in replication defects and cellular replication stress sensitivity, but these phenotypes can
be reversed by a Pol31 gain-of-function mutation that enhances the stability of the Pol3-Pol31
complex.26,27 Conversely, a Pol31 polymorphism that destabilizes the interaction of Pol31 with
Pol3 renders POL32 essential in budding yeast.28 Importantly, the Pol \u03b4-stabilizing Pol31 mutation
does not suppress the profound BIR defect caused by loss of Pol32 (ref. 26), highlighting BIR-
specific functions uniquely associated with Pol32. Consistently, BIR efficiency is negatively
affected by disruption of PCNA and DNA polymerase \u03b1 interaction sites within the Pol32 C-
terminal domain (CTD) spanning aa 116-350 (ref. 23). Yet, how Pol32 orchestrates BIR efficiency
remains to be fully investigated.

Here, we identify an RBM within the Pol32 CTD. Its phosphorylation enhances Pol32
RBM-RPA interactions, providing the first example of phospho-activated binding to the Rfa1 basic-
hydrophobic groove. Corresponding phospho-mimetic amino-acid substitutions boost BIR
efficiency in cells. Thus, Pol32 is not a mere enabler of BIR, but serves as an acceptor of post-
translational modifications that cells can utilize to dynamically regulate BIR efficiency through
modulating the affinity of Pol \u03b4 for RPA.

Results
A Pol32 C-terminal element promotes BIR. Pol32 is 350 aa in length. The Pol32 CTD is not
visible in deposited EM structures25, adopts an elongated shape29, and is predicted by
AlphaFold30-32 to be largely disordered (Fig. 1A). A C-terminus-proximal PCNA-interacting protein-
box (PIP-box)33 and a DNA polymerase \u03b1 interaction motif (DPIM)34,35 spanning aa 270-309 have
previously been identified within the Pol32 CTD. By complementing pol32\u2206 cells with a series of
plasmid-borne Pol32 mutants, it has been shown that BIR efficiency is reduced by disruption of
the Pol32 PIP-box and large internal deletions that affect the DPIM while leaving the PIP-box
intact.23 To better understand how Pol32 promotes BIR, we truncated Pol32 from the C-terminus
in a stepwise manner (Fig. 1B). The truncations were introduced at the endogenous POL32 locus,
and the truncated proteins were expressed at levels comparable to the wild-type protein (Fig. 1C).
The impact of these mutations on BIR was determined using a reporter strain23 for BIR efficiency
at a DSB on chromosome V with the only available repair template located at an ectopic site on
chromosome XI (Fig. 1D). The DSB is effectively introduced across the cell population upon
galactose-induced expression of the HO endonuclease23 (Supplementary Fig. 1A). BIR-mediated
DSB repair can be genetically tracked as it restores a CAN1 marker at the cut site whilst leading
to the loss of a strategically placed HPH marker (Fig. 1D and Supplementary Fig. 1B). Confirming
earlier work23,33, the PIP-box truncation (Pol32_1-333) disrupted Pol32-PCNA interactions in yeast
two-hybrid assays (Supplementary Fig. 1C) and resulted in a reduction (~5-fold) in BIR efficiency
compared to cells harbouring wild-type POL32 (Fig. 1E). Further truncation of endogenous Pol32
to Gln269, thereby removing the DPIM and disrupting Pol \u03b1 interactions (Supplementary Fig. 1D),
caused a further ~2-fold reduction in BIR efficiency (Fig. 1E). These observations are consistent
with the notion that interactions with both PCNA and Pol \u03b1 are important for the role of Pol32 in
BIR23. However, removal of a further 20 aa (Pol32_1-249) was required to reduce BIR efficiency
to the level seen in pol32\u2206 cells (Fig. 1B and E). Next, we analysed DSB repair in an independent
tester strain which is disomic for chromosome III (with one copy harbouring an HO-cut site and
the other serving as repair template)36. In this system, DSB repair is highly efficient, and cells
overwhelmingly use BIR with a small contribution of DSB repair by gene conversion. In contrast,
pol32\u2206 cells show reduced BIR efficiency, an increase in chromosome loss events, and the
formation of half-crossovers indicative of failed DSB repair and aberrant processing of BIR
intermediates, respectively.36 We observed similar levels of Pol32-independent BIR, chromosome
loss, and half-crossovers in cells harbouring a POL32 deletion or a truncation of Pol32 at residue
249 (Supplementary Fig. 1E and F). Thus, Pol32_1-249 results in a Pol32-null phenotype for
ectopic and allelic interhomolog BIR. In contrast, expression of Pol32_1-249 does not cause
cellular replication stress or cold sensitivity, which are the phenotypes that signify replication
defects associated with complete loss of Pol32 and Pol3-Pol31 instability (Fig. 1F).
Accordingly, Pol32_1-249 is a separation-of-function mutant, showing that the C-terminal
~100 aa of Pol32 are required for BIR in their entirety, whilst being dispensable for canonical
replication functions, which depend upon Pol \u03b4 stability. Moreover, this region contains a functional
element impacting BIR, approximately spanning aa 250 to 269 within the Pol32 CTD.

Pol32 contains an RBM and mediates Pol \u03b4-RPA interactions. AlphaFold predicts a small \u03b1-
helical feature spanning Pol32 aa 244 to 261 (marked as Helix in Fig. 1A) that overlaps with the
aforementioned functional element relevant for BIR efficiency. Its sequence
(KNNDDLEDLLETTAEDS) is rich in acidic and hydrophobic residues, which are segregated to
opposite faces of the \u03b1-helix. Such amphipathic helices are often involved in protein-protein
interactions and can be found in RBMs of established RPA client proteins.13-19 Typically, acidic-
hydrophobic RBMs engage in interactions with a basic-hydrophobic groove found within the N-
terminal OB domain (also known as DBD-F) of Rfa1/RPA70; this has been demonstrated in yeast
by structural analyses of the Rfa1 NTD in complex with an RBM found in Ddc2 (the integral partner
of the cell cycle checkpoint kinase Mec1).16,37 To address the possibility that Pol32 may harbour
a hitherto unidentified RBM, we used AlphaFold38 to generate structural models exploring a
potential interaction between Pol32 and Rfa1. Here, a C-terminal section of full-length Pol32 was
consistently predicted to associate with the Rfa1 NTD, as supported by analysis with
AlphaBridge39 (Supplementary Fig. 2A). Subsequent truncation of the input sequence to include
just aa 237-267 of Pol32 produced highly consistent models (Supplementary Fig. 2B). These
show that aa 241-263 of Pol32 adopt an amphipathic \u03b1-helical conformation that occupies the
groove of Rfa1 (Fig. 2A). However, the trajectory and position of the putative Pol32 RBM differs
from Ddc2; it binds with opposite polarity and does not have a kink in its path when bound to Rfa1
(Fig. 2A). Three hydrophobic residues, Leu250, Leu253 and Leu254 (coloured teal, green and
orange, respectively) serve to anchor Pol32 into the hydrophobic section of the Rfa1 groove (lined
by residues Leu47, Leu60, Ala88, Val97, and Leu99, see Fig. 2A and Supplementary Fig. 2C). By
structural comparison, the side chains of these three residues have equivalency to Ile17, Leu18
and Leu21 of Ddc2 (Figure 2A and Supplementary Fig. 2D; PDB: 8B4J). Reversal of the amino
acid sequence of Ddc2 (reading it from the C- to the N-terminus) allows a simpler comparison
with Pol32 and suggests that hydrophobic residues at these positions are conserved, as part of
an \u03d5-D/E-x-\u03d5-\u03d5 motif (where \u03d5 denotes a hydrophobic and x any amino acid residue). We note
that a semi-conserved region of amino acids follows (or precedes) this motif, consisting of either
negatively charged amino acids, and/or several serine and threonine residues. Within this region,
the side chain of Rfa1 Arg44 is poised to interact with Pol32 Asp260, whereas for Ddc2 it sits in
proximity to the backbone carbonyl of Ser11 (see Supplementary Fig. 2C and D); as Ser11 of
Ddc2 is known to be phosphorylated, this immediately suggests that additional negative charge
can be added to this patch via post-translational modification.
Next, we sought to determine whether Pol32 can mediate RPA interactions in the context
of the Pol \u03b4 holoenzyme. Affinity capture of endogenously TAP-tagged Pol31 co-precipitated the
remaining subunits of Pol \u03b4, Pol3 and Pol32, from whole cell extracts (Supplementary Fig. 3A).
We found that RPA was enriched in Pol \u03b4 precipitates. However, an internal deletion of Pol32 aa
247 to 258 (hereafter referred to as \u2206RBM) diminished Pol \u03b4-RPA interactions (Fig. 2B). These
data are therefore consistent with the acidic-hydrophobic motif within Pol32 functioning as a bona
fide RBM and serve to confirm the identified Pol \u03b4-RPA interaction.
To measure the affinity of the Pol32 RBM for RPA, we synthesized a fluorescein-labelled
peptide derived from Pol32 aa 243 to 264 and tested binding to purified Rfa1 NTD (aa 1-132)37
using fluorescence polarization (FP). We determined a KD of 22.9 \u03bcM for the interaction (Fig. 2C).
This is commensurate with affinities that have been determined for other RBM-RPA interactions.14
As expected, targeting key hydrophobic interactions indicated by the Pol32 RBM-Rfa1 model by
introducing mutations Leu253Arg and Ala258Arg into the Pol32 RBM (hereafter referred to as
RBM*) dramatically reduced the apparent binding affinity for the Pol32-derived peptide (KD not
determined; see Fig. 2C).
Perhaps surprisingly, neither the \u2206RBM nor the RBM* mutation in the context of full-length
Pol32 had apparent effects on BIR efficiency in vivo (Fig. 2D). However, when the RBM was
mutated together with the PIP-box (Pol32_1-333 RBM*), cells exhibited a pol32\u2206-like BIR
phenotype (Fig 2D), while retaining replication competence (Supplementary Fig. 3B). Thus, the
Pol32 PIP-box and RBM can function independently to promote BIR efficiency.
A PCR-based BIR assay23 for the detection of new DNA synthesis initiated at the
centromere-proximal side of the HO cut site (see Supplementary Fig. 1B) upon invasion of the
repair template showed that Pol32 RBM* was associated with a slight but reproducible delay in
the formation of BIR repair intermediates in the first 8 h after DSB induction. While the levels of
BIR product formation mediated by Pol32 RBM* were eventually indistinguishable from wild-type,
Pol32_1-333 RBM* was associated with a prolonged BIR DNA synthesis defect that grew more
pronounced between 8 and 20 h after DSB induction (Fig 2E). Together, these data indicate that
Pol32 RBM-RPA interactions promote BIR.

The Pol32 RBM is phosphorylated by multiple protein kinases. The newly identified Pol32
RBM contains three potential phosphorylation sites at Thr256, Thr257, and Ser261. Phospho-
proteomic experiments have shown that Pol32 can be phosphorylated at all three sites in vivo.40-
44 Whilst fully phosphorylated RBM peptides were observed in a subset of these studies42-44,
phosphorylation at Thr257, either alone or in combination with phosphorylation at Thr256, was
more common. Notably, phosphorylation at Ser261 alone was not reported. Thus, the Pol32 RBM
appears to be predominantly modified at both threonine residues. While the function and
mechanism of Pol32 RBM phosphorylation remain to be determined, phospho-proteomic
evidence suggests the involvement of multiple kinases in vivo. These include the acidophilic Dbf4-
dependent kinase (DDK) Cdc7 (ref. 44), cyclin-dependent kinase (CDK) Cdc28 (ref. 40), and
Polo-like kinase (PLK) Cdc5 (ref. 42). To test direct phosphorylation by these kinases, we purified
a recombinant Thioredoxin-Pol32 fusion protein, incorporating Pol32 aa 248-264 (Trx-RBM), and
subjected it to in vitro kinase reactions with TAP-tagged versions of Cdc7, Cdc28 (as well as
Cdc28-associated cyclins Clb1, 3, and 5), and Cdc5 (as well its downstream kinase Cdc15)
(Supplementary Fig. 4A). We also tested the acidophilic casein kinase CKII (Cka2-TAP) and
GSK3 kinase ortholog Rim11-TAP, which have predicted45 consensus phosphorylation target sites
at Thr256 and Thr257, respectively. Phosphorylation of Trx-RBM was observed with affinity-
captured Cdc7, Cdc5, and CKII, but not Cdc28 or Rim11. The levels of Trx-RBM phosphorylation
mediated by Cdc7, Cdc5, and CKII were visibly diminished when a fusion protein with Pol32
T256A and T257A substitutions was used in the kinase assay (Supplementary Fig. 4B), indicating
that at least part of the observed kinase activity was directed at the Thr256/257 twin
phosphorylation site. Next, we mapped phosphorylation events using mass spectrometry (Fig.
3A), which confirmed the ability of affinity-captured Cdc5, CKII, and Cdc7 to phosphorylate Pol32
RBM residues Thr256, Thr257, and Ser261 in vitro (Fig. 3B and Supplementary Fig. 4C). Here,
the greatest number of Pol32 RBM phospho-peptides were generated by Cdc7, which has
previously been implicated46 in the regulation of BIR. In addition, these data confirmed the DDK
and PLK-dependent phosphorylation of the Pol32 RBM, which has been observed in vivo, and
suggest that Cdc5, Cdc7, and possibly additional acidophilic kinases such as CKII, may contribute
to establishing its phosphorylation.

Pol32 RBM phosphorylation boosts RPA affinity and BIR. To address the impact of Pol32
RBM phosphorylation on RPA interactions, we synthesized different versions of the fluorescein-
labelled Pol32 RBM peptide and tested interaction with the Rfa1 NTD by FP as before. Here, the
pThr257 peptide bound with a KD of 4.3 \u03bcM, a ~5-fold increase in affinity compared to the non-
phosphorylated version of the Pol32 peptide. The bis-phosphorylated Pol32 pThr256 pThr257
peptide bound with a KD of 0.14 \u03bcM, an additional ~30-fold increase in affinity over the Pol32
peptide phosphorylated at Thr257 only (and a ~160-fold increase compared to the
unphosphorylated peptide). Finally, additional phosphorylation at Ser261 did not have a marked
effect on affinity with a Pol32 pThr256 pThr257 pSer261 peptide binding to Rfa1 NTD with a KD
of 0.11 \u03bcM (Fig. 3C). Thus, Pol32 RBM bis-phosphorylation at the Thr256/257 twin site
significantly strengthens Pol32-RPA interactions.
To rationalise enhanced RPA binding upon Pol32 RBM phosphorylation, we used the
functionality of AlphaFold 3 to model the interaction of the phosphorylated RBM (pRBM) with Rfa1
(Fig. 3D and Supplementary Fig. 5). This suggests that phosphorylation of Thr257 breaks the
extended alpha-helical conformation that was predicted for the non-phosphorylated Pol32 RBM
(see Fig. 2A), positioning the phosphate so that it sits in proximity to the side chains of Rfa1 Arg91
and -Lys95, and generating a kink in the modelled path of the bound peptide. We validated the
importance of this modelled set of contacts by introducing Arg91Ala and Lys95Ala substitutions
into Rfa1 NTD (Rfa1-MUT) and then testing interactions by FP. Here, the previously observed
enhancement of binding driven by phosphorylation of Thr257 in the Pol32 RBM was neutralized
(Supplementary Fig. 6A).
Within the short helical element immediately following pThr257 (aa 258-262, AEDSL), the
side chain of Ala258 packs against the face of Leu262 and projects down into a hydrophobic
pocket formed by Rfa1 Met49, -Leu47 and the side chain carbon atoms of Rfa1 Asn33 and -Lys58
(Fig. 3D and Supplementary Fig. 5B). Hydrophobic interactions with this so-called side-pocket
have previously been observed for RPA client proteins engaged with the Rfa1 NTD hydrophobic
groove and serve to enhance the overall interaction strength.14 This suggests an induced-fit
mechanism where Pol32 RBM phosphorylation at Thr257 facilitates a conformational change that
allows higher-affinity Rfa1 NTD interactions. In line with the further enhanced affinity of the
Thr256/257 bis-phosphorylated RBM for Rfa1 NTD measured in vitro, our modelling also
suggests that the additional phosphorylation of Thr256 would be tolerated, picking up potential
hydrogen bonds with the side chains of Rfa1 Arg35 and -Arg44. Modelling of the bis-
phosphorylated Pol32 RBM showed that the phosphorylated threonines swap partners,
presumably to eliminate torsional stress from the polypeptide backbone, but continue to interact
with the same basic residue pairings of Arg91/Lys95 and Arg35/Arg44 (Fig. 3D and
Supplementary Fig. 5C). In agreement with this hypothesis, the binding affinity of the
pThr256/pThr257 peptide was reduced to that of the mono-phosphorylated peptide when titrations
were carried out with Rfa1-MUT (Supplementary Fig. 6B). Our models indicate that the short helix
that follows the phosphorylation sites would be slightly shifted in register (aa 259-263, EDSLM)
with Leu262 still anchored in the hydrophobic side-pocket, but with Ala259 now displaced (Fig.
3D and Supplementary Fig. 5D).
Finally, whilst models of the fully phosphorylated Pol32 RBM bound to Rfa1 NTD were of
poorer quality (lower pLDDT (predicted Local Distance Difference Test) score and consistency),
they indicated that that pSer261 may point towards the solvent and make no apparent interaction
with Rfa1 NTD. This is consistent with our biophysical data, where phosphorylation of Ser261
makes a negligible contribution to the overall affinity (Fig. 3C and Supplementary Fig. 5E, F). We
therefore conclude that it is phosphorylation at the two sequential threonines 256/257 of the Pol32
RBM that drives a high-affinity interaction with Rfa1 NTD.
Addressing the impact of enhanced Rfa1 NTD interactions mediated by Pol32 pRBM on
BIR, we confirmed that phospho-mimetic amino changes Thr256Asp and Thr257Asp within the
RBM increase the affinity for Rfa1 (Supplementary Fig. 6C). We then generated versions of the
ectopic BIR reporter strain expressing Pol32 T256D T257D, which was present at concentrations
comparable to wild-type Pol32 (Fig. 3E). Importantly, Pol32 T256D T257D boosted BIR efficiency
as compared to the control strain (Fig. 3F). Like Pol32 \u2206RBM and -RBM*, an endogenously
expressed Pol32 T256A T257A phospho-null mutant was not associated with a BIR efficiency
defect (Fig. 3F). However, as in the case of Pol32_1-333 RBM*, a strain expressing Pol32_1-333
T256A T257A showed a significant drop in BIR efficiency, suggesting that phosphorylation at
Thr256/257 is required for BIR efficiency in the absence of the Pol32 PIP-box. Consistently,
T256D/T257D continued to enhance BIR efficiency in the context of a Pol32 PIP-box truncation
mutant (Fig. 3G). These data are in line with the notion that the phosphorylation of the Pol32 RBM
in vivo40-44 can be deployed by cells to modulate BIR efficiency.

Pol32 pRBM, Pif1, and Mph1 similarly target Rfa1. Recently, an RBM has been identified within
the accessory BIR helicase Pif1 and shown to be required for BIR efficiency.20 Mph1, which
functions as an antagonist of BIR by dissociating extended D-loops47-49, has also been shown to
bind RPA50,51, although the details of this interaction and its potential significance to BIR is not
known. Inspection of the Mph1 C-terminus revealed an acidic-hydrophobic amino acid sequence
(residues 981-993) with close resemblance to the Pol32 RBM. While the Pif1 RBM has been
defined as spanning aa 60 to 74 (ref. 20), Trp75 within Pif1 aligns well with a similar hydrophobic
residue, Phe993, at the end of the putative RBM sequence in Mph1 (Fig. 4A). Interestingly, both
Pif1 and Mph1 RBMs contain pre-existing negative charges (Asp71 and Asp990, respectively) at
the position of the Thr256/257 double phosphorylation site within Pol32, suggesting that both
RBMs might behave more similarly to the phosphorylated rather than the non-phosphorylated
Pol32 RBM. Indeed, AlphaFold modelling predicts that the Pif1 and Mph1 RBMs bind Rfa1 with
the same polarity as Pol32, placing three hydrophobic residues consistent with the \u03d5-D/E-x-\u03d5-\u03d5
motif into the Rfa1 groove: Pif1 Leu66, -Leu68 and -Leu69; Mph1 Leu983, -Ile986 and -Leu987
(Fig. 4A and Supplementary Fig. 7A, B). Pif1 Trp75 and Mph1 Phe993 bind directly into the
hydrophobic side-pocket that is occupied by Leu262 in the phosphorylated forms of the Pol32
RBM (Supplementary Fig. 7C, D). Here, the additional phosphorylation of the Pif1 or Mph1 RBMs
is not predicted to significantly impact the position of Pif1 Trp75 and Mph1 Phe993, which
suggests that both proteins adopt the high-affinity Rfa1 binding confirmation independently of
phosphorylation within their RBMs (Supplementary Fig. 7E, F). Consistently, we determined a KD
of 0.32 \u03bcM for a fluorescein-labelled peptide derived from Pif1 aa 59 to 79 with the Rfa1 NTD by
FP (Fig. 4B). Thus, Pif1 tightly binds RPA and with similar affinity to Pol32 RBM phosphorylated
at Thr256/257. Phosphorylation of Pif1 at Ser70 and Ser72 flanking Asp71 had a moderate impact
on Rfa1 NTD binding affinity; improving the KD to 0.15 \u03bcM (Supplementary Fig. 8A). Phospho-null
(Ser70Ala/Ser72Ala) or phospho-mimetic (Ser70Asp/Ser72Asp) amino acid substitution in
endogenous Pif1 had no significant effect on BIR efficiency in vivo (Supplementary Fig. 8B).
However, consistent with a reported dependence of BIR efficiency on the Pif1 RBM20, an internal
deletion of the Pif1 RBM resulted in a significant BIR defect (Fig. 4C).
To validate the Mph1 RBM, we synthesized a fluorescein-labelled peptide derived from
Mph1 residues 972 to 993 and determined a KD of 2.09 \u03bcM with Rfa1 NTD by FP (Fig. 4D). Thus,
like Pif1, Mph1 contains an RBM with an intrinsic affinity to Rfa1 similar to phosphorylated
versions of the Pol32 RBM. As expected, an FP competition experiment between the Mph1 and
Pol32 RBM peptides showed that binding is mutually exclusive, confirming that both proteins
target the Rfa1 NTD basic-hydrophobic groove (Supplementary Fig. 8C). To test whether RPA
interactions may be required for the role of Mph1 in BIR, we truncated the protein to remove the
39 C-terminal aa that have been shown50 to be required for interactions with RPA. Cells
expressing the truncated Mph1_1-954 from the endogenous locus exhibited significantly elevated
BIR efficiency, phenocopying a complete deletion of MPH1 (Fig. 4E). Consistently, BIR
intermediate formation was higher in mph1_1-954 cells compared to MPH1 wild-type control cells
as measured by PCR23 6 h after DSB induction (Supplementary Fig. 8D). In contrast, Mph1_1-
954 was not associated with the spontaneous mutator phenotype52 that is caused by loss of Mph1
(Supplementary Fig. 8E). These results demonstrate that Mph1_1-954 remains active but loses
the ability to limit BIR efficiency by D-loop dissociation. Finally, we mutated Mph1 Phe993 to
alanine and found that this single amino acid change results in significantly elevated BIR
efficiency, phenocopying Mph1_1-954 (Fig. 4E). We conclude that the high-affinity RPA binding
mode which relies on RBM contacts within the Rfa1 NTD basic-hydrophobic groove and the
hydrophobic side-pocket (ref. 14) is critical for Mph1 to engage and disassemble D-loop
intermediates during BIR.
Overall, these findings show RBM-mediated RPA interactions of each BIR replisome
component and BIR antagonist Mph1 impact homologous recombination-dependent DNA
synthesis, highlighting a critical scaffolding function of RPA bound at recombination intermediates.
Pif1 and Mph1 intrinsically bind RPA with high affinity, whereas phosphorylation is required to
activate high-affinity RPA binding by the Pol32 RBM, providing a mechanism for the dynamic
regulation of BIR efficiency (Fig. 4F).

A model for the actions of Pol32 in BIR. The existing cryo-EM structure of the budding yeast
Pol \u03b4-PCNA-DNA complex does not resolve the Pol32 CTD53. We therefore modelled full-length
Pol32 bis-phosphorylated at Thr256/257 as part of the Pol \u03b4-PCNA-DNA complex and included
the Rfa1 NTD (Fig. 5A and Supplementary Fig. 9A). As expected, modelling shows that the Pol \u03b4
catalytic subunit Pol 3 employs a noncanonical PIP-box motif embedded in the cysteine-rich
metal-binding motif A (CysA/PIP) to bind one subunit of the trimeric PCNA DNA clamp;
recapitulating features of the published structure. Pol31 binds to Pol3 and is in turn bound by the
Pol32 NTD. A helix-turn-helix motif (which we refer to as Hook; Pol32 aa 164-218) within the Pol32
CTD bound to one monomer-monomer junction of the PCNA homotrimer (Fig. 5B). This mode of
interaction is distinct from that of a canonical PIP-box with the PCNA interdomain connector loop
(IDCL), but resembles that previously described54 for a helix-turn-helix motif within translesion
polymerase Rev1 and PCNA. The Pol32 Hook may provide another PCNA anchor point for Pol32
but is not sufficient to support detectable PCNA interactions or BIR efficiency (see Fig. 1E and
Supplementary Fig. 1C). Interestingly, the Pol32 PIP-box is consistently engaged with a PCNA
IDCL in our models. This suggests that the Pol32 CTD is sufficiently flexible to allow the Pol32
PIP-box to reach one of the two PCNA IDCL binding sites not occupied by Pol3 (Supplementary
Fig. 9B). Importantly, we observed a consistently predicted interface between the phosphorylated
Pol32 RBM and the Rfa1 NTD in the context of the entire Pol \u03b4-PCNA-DNA-Rfa1 NTD assembly
(Fig. 5A and Supplementary Fig. 9A). Moreover, the conformational flexibility offered by the length
of the intervening linker sequence between the Rfa1 NTD and the subsequent RPA subdomains,
as well as that of the Pol32 CTD itself, predicts that Pol32 could interact with RPA bound to a
displaced ssDNA strand at a D-loop, even while Pol \u03b4 is engaged with PCNA at the primer junction
during recombination-dependent DNA synthesis (Fig. 5C). As expected, there is no consistent
positioning or interaction of the Pol32 DPIM with the Pol \u03b4-PCNA-DNA-Rfa1 NTD assembly.
This is in line with the requirement for the DPIM for interactions of Pol32 with Pol \u03b1 as shown by our
yeast-two-hybrid analysis (Supplementary Fig. 1D) and recently published55 binary AlphaFold
models of the Pol32 DPIM-Pol \u03b1 interaction. Pol32 DPIM-Pol \u03b1 interactions come into play late in
the BIR process, when the Pol \u03b1-primase complex initiates lagging strand synthesis on the
nascent strand.22,56-58 Pol32-Pol \u03b1 interactions may then support the exchange for Pol \u03b4 and
enhance BIR efficiency by promoting the conversion of the nascent ssDNA into duplex DNA.23,24
Taken together, our model is consistent with the independent contributions which we have
observed the Pol32 RBM and PIP-box make to BIR efficiency. We propose that multi-point
interactions of Pol32 and Pif1 (refs. 20,59) with PCNA and RPA-bound recombination
intermediates ensure the proper recruitment and retention of Pol \u03b4 and the accessory D-loop
helicase Pif1 at BIR intermediates, allowing optimal BIR reactions to take place (Fig. 5D).

Methods
Yeast strains
All S. cerevisiae strains used in this study were derived from BY4741 (ref. 67), JRL092 (ref. 23),
AM1003 (ref. 36), or pJ69-4A (ref. 68) and are described in Supplementary Table 1. Yeast was
grown at 30\u00b0C, or the indicated temperatures, in rich (YP) or minimal media supplemented with
either glucose (D), galactose (GAL), or raffinose (RAFF) to a final concentration of 2% (w/v). For
solid media, agar was added to a final concentration of 1% (w/v).

Yeast two-hybrid assay
For yeast two-hybrid experiments69, strain pJ69-4A was co-transformed with vectors pGBKT7 and
pGADT7, each either unmodified or modified to express protein constructs fused to the Gal4-
binding or Gal4-activating domain. Logarithmic cultures of the respective strains (Supplementary
Table 1) grown in minimal medium lacking tryptophan and leucine were diluted and spotted on
control plates (minimal medium lacking tryptophan and leucine) and selective plates (minimal
medium lacking tryptophan, leucine, and histidine) containing the histidine biosynthesis inhibitor
3-amino-1,2,4-triazole (3-AT). The interaction between GBD-Stn1 and Ten1 with a GAD-Pmt3-
Tpz1 fusion70 was used as a positive control.

BIR efficiency measurements
To measure ectopic BIR (JRL092-derived strains), logarithmically growing cells cultured in
YPRAFF medium were plated in appropriate dilutions onto YPD or YPGAL medium and grown
into colonies. Cell viability as a measure of DSB repair efficiency after HO endonuclease induction
was derived by dividing the number of colony-forming units (CFU) on YPGAL by that on YPD. For
JRL-derived strains, HO cut efficiency was routinely tested by PCR, and a PCR product spanning
the HOcs locus was no longer obtained after exposing cells to galactose for 1 h (for an example,
see Supplementary Fig. 1A). Colonies on YPGAL were always replica plated onto YPD medium
supplemented or not with 200 \u03bcg/ml hygromycin B. This showed that 2-4% of DSB repair events
led to retention of the HPH resistance marker, in good agreement with previously determined23
rates of BIR-independent DSB repair by gene conversion in JRL-derived strains. This fraction was
subtracted from the value for DSB repair efficiency to calculate BIR efficiency for a given strain.
In addition, BIR survivors were routinely validated using PCR on random colonies to show the
presence of a newly formed, contiguous CAN1 marker at the former HOcs locus (see
Supplementary Fig. 1B).
To measure allelic BIR (AM1003-derived strains), logarithmically growing cells cultured in
YP-raffinose medium were plated in appropriate dilutions onto YPD or YPGAL medium. Colonies
on YPGAL were replica-plated onto minimal media lacking adenine and leucine, leucine only, and
lacking leucine whilst containing limited amounts of adenine (5 mg/l) to determine the contribution
of different DSB repair pathway to cell survival: ADE+ leu- colonies arise by BIR, ADE+ LEU+
colonies by GC; half-crossover formation and chromosome loss give rise to white ade3- leu- and
red ade1- leu- colonies on minimal media supplemented with limited amounts of adenine,
respectively.
Every strain was tested at least three times and differences in mean values for BIR
efficiency were assessed for significance using Welch\u2019s t-test.

BIR kinetics measurements
PCR analysis in JRL-derived strains was performed on genomic DNA prepared such that final
DNA concentrations were equal across samples.23 HOcs cleavage was confirmed by amplifying
genomic DNA with primers P3 (5'-TTGCATGGTTATTTATCTCAATCTC) and P4 (5'-
ACGTCAAGACTGTCAAAGGA). BIR intermediates were amplified with primers P1 (5'-
TTTCCCGACGAGAGTAAATGGCGA) and P2 (5'-GGTGCAAAAGCCGTGAAACC) (see Fig 1D)
using Phusion polymerase (NEB). PCR products were run on 0.9% agarose gels in 0.5 x TBE
buffer with ethidium bromide and quantified (signal to noise ratio) using a Gel Doc imaging system
(Syngene Ingenius LHR). Ratios were divided by those determined for a control amplified from
an independent locus (CDC5; using primers 5'-GGTGAAGGTGGATTTGCTC and 5'-
ATGTCCAGAATGCTTACCCAT) on chromosome XIII from the same input and under the same
conditions and normalized to the value obtained with wild-type POL32 at 21 h. Uncropped gels
are provided within the Source Data file.

Immunoprecipitation
Yeast cell cultures were harvested at 4\u00b0C and washed sequentially with an ice-cold solution of 20
mM HEPES-KOH (pH 7.9) and a wash buffer containing 100 mM HEPES-KOH (pH 7.9), 50 mM
potassium acetate, 10 mM magnesium acetate and 2 mM EDTA. Cells were then centrifuged and
resuspended in wash buffer supplemented with 2 mM sodium glycerophosphate (Johnson
Matthey), 2 mM sodium fluoride (Fisher Scientific), 1 mM DTT, 1% (v/v) protease inhibitor cocktail
for fungal and yeast extracts (Sigma), and 0.24% (w/v) EDTA-free complete protease inhibitor
cocktail (Roche). The mixture was flash frozen in liquid nitrogen and protein immunoprecipitation71
was carried. In brief, cells were lysed using a mechanical pestle and mortar (Cole-Parmer SPEX
Sample Prep 6850) pre-cooled at -80\u00b0C. To each gram of thawed lysate, 0.25 ml of a solution of
50% (v/v) glycerol, 100 mM HEPES-KOH (pH 7.9), 50 mM potassium acetate, 20 mM magnesium
acetate, 0.5% Igepal CA-630, 2 mM EDTA supplemented with 2 mM sodium glycerophosphate, 2
mM sodium fluoride, 1 mM DTT, 1% (v/v) protease inhibitor cocktail for fungal and yeast extracts
(Sigma), and 0.24% (w/v) EDTA-free complete protease inhibitor cocktail was added. Pierce
universal nuclease was added to a final concentration of 1.6 U/\u03bcL, and samples were agitated on
a rotating platform at 4\u00b0C for 40 min. Lysates were then clarified by successive centrifugations at
4,000 g and 13,000 g.
50 \u03bcL analytical samples of the supernatant were mixed with 1.5 x Laemmli buffer (100 \u03bcL)
and boiled for four minutes (Input samples). The remaining supernatant was incubated with 100
\u03bcL TAP-beads (M270 Dynabeads epoxy beads coupled with anti-sheep IgG) at 4\u00b0C for 2 h. The
beads were then washed three times with 100 mM HEPES-KOH (pH 7.9), 50 mM potassium
acetate, 20 mM magnesium acetate, 2 mM EDTA, 0.1% (v/v) Igepal CA-630. The first wash was
supplemented with 2 mM sodium glycerophosphate, 2 mM sodium fluoride, 1 mM DTT, 1% (v/v)
protease inhibitor cocktail for fungal and yeast extracts (Sigma), and 0.24% (w/v) EDTA-free
complete protease inhibitor cocktail (Roche). After washing, the beads were mixed with 100 \u03bcL 1
x Laemmli buffer and boiled for four minutes (IP samples). Input and IP samples were analysed
by western blotting.

Protein kinase assays
TAP-tagged kinases were immunoprecipitated as described above except for lysate
resuspensions and wash buffers containing 300 mM potassium acetate. TAP beads were then
washed twice with 20 mM Hepes-KOH (pH 7.9), 150 mM NaCl. Radioactive kinase assays: the
beads were mixed with 75 \u03bcM of peptide substrate, 0.625 mM of ATP and 1.5 \u03bcL of ATP [ \u03b3-32P]
(6000Ci/mmol) in a buffer of 20 mM Hepes-KOH (pH 7.9), 150 mM NaCl, 10 mM MgCl2 and 0.5
mM TCEP and incubated in a 30\u00b0C water bath. The supernatant containing the phosphorylation
substrate was then mixed with 1/3 volume of 3 x Laemmeli buffer and boiled for 5 min. Samples
were resolved by SDS-polyacrylamide gel electrophoresis and the gels were dried under vacuum
at 80\u00b0C (Gel Dryer Model 583, BioRad). The dried gels were exposed to phosphor-imaging plates
and imaged using a Typhoon Imager (Amersham). Uncropped gels and phosphor images are
provided within the Source Data file.
For non-radioactive kinase assays followed by mass spectrometric analysis, the TAP
beads were mixed with 75 \u03bcM of peptide substrate and 2.5 mM of cold ATP in a buffer of 20 mM
Hepes-KOH (pH 7.9), 150 mM NaCl, 10 mM MgCl2, 1 x PhosSTOP (Merck)and 0.5 mM TCEP,
before incubation in a 30\u00b0C water bath. After 2 or 24 h, the supernatant containing the
phosphorylation substrate was immediately frozen at -20\u00b0C.

TCA protein extraction
Logarithmically growing cells were harvested by centrifugation, washed in deionised water, and
resuspended in 300 \u03bcL 20% (w/v) trichloroacetic acid (TCA). After adding a small quantity of acid-
washed glass beads, cells were lysed by shaking vigorously thrice for one minute, with 20
seconds of cooling on ice between shaking cycles. The supernatant was recovered and combined
with a subsequent wash of the remaining cell debris and glass beads with 300 \u03bcL 5% (w/v) TCA.
Proteins were pelleted by centrifugation at 4,000 g for 10 min and resuspended in 100-200 \u03bcL of
1 x Laemmli buffer supplemented with 150 mM Tris base. Samples were boiled for four minutes
and cleared by centrifugation at 4,000 g for 10 min before western blotting.

Western blotting
Protein samples were resolved by SDS-polyacrylamide gel electrophoresis on 5-10%
polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were incubated
in TBST containing 5% (w/v) skimmed milk and one of the following antibodies at the indicated
dilutions: anti-TAP consisting of peroxidase anti-peroxidase soluble complex antibody produced
in rabbit (Sigma P1291, 1:5,000), mouse anti-Myc (Santa-Cruz sc-40, 1:1,000), mouse anti-FLAG
(Sigma F3165, 1:5,000), rabbit anti-Rfa1 (Abcam ab221198, 1:3,000). After three washes in
TBST, the relevant HRP-conjugated secondary antibody was added (Cell Signaling Technology
goat-anti-rabbit 7074, 1:10,000; Cell Signaling Technology horse-anti-mouse 7076, 1:10,000).
Finally, membranes were washed thrice in fresh TBST, treated with western blotting reagents, and
chemiluminescence was captured using an ImageQuant 800 (Cytiva) western blot imaging
system. Uncropped blots are provided within the Source Data file.

Recombinant protein
For isolation of Rfa1 NTD protein37 corresponding to amino acids 1 to 132, a 6xHis-GST-Rfa1
NTD construct was expressed overnight in E. coli BL21 (DE3) cells in a shaking incubator set at
180 rpm and 20\u00b0C. Cells were harvested by centrifugation and frozen at -80\u00b0C. After resuspension
in a buffer containing 50 mM HEPES, 150 mM NaCl, 0.5 mM TCEP (pH 7.5), cells were lysed by
sonication, and cell lysates were clarified by centrifugation. 6xHis-GST-Rfa1 NTD was affinity-
captured using Ni Sepharose (Cytiva), eluted using imidazole, then applied to a size exclusion
chromatography column, equilibrated in 20 mM HEPES (pH 7.5), 150 mM NaCl at 25\u00b0C. Rfa1
NTD (>98% purity) was concentrated, flash frozen in liquid nitrogen, and stored at -80\u00b0C. The
Rfa1 NTD Arg91Ala and Lys95Ala double mutant (Rfa1-MUT) was expressed and purified in a
similar manner, except that Co Sepharose (Cytiva) was used.

Peptides
His6-tagged Trx-RBM fusions were expressed in E. coli strain BL21(DE3). Transformed bacteria
were cultured in Turbo Broth (Molecular Dimensions) supplemented with 50 \u03bcg/ml carbenicillin,
at a temperature of 37\u00b0C, in an orbital-shaking incubator set at 200 rpm. Recombinant protein
expression was induced by the addition of 0.2 mM isopropyl \u03b2-d-1-thiogalactopyranoside once
the optical density at 600 nm had reached 1.5. The temperature of the incubator was then reduced
to 20\u00b0C, and the cultures grown for an additional period of ~16 hours. After this, cells were
harvested by centrifugation at 4000 g for 20 minutes at 4\u00b0C and then stored at -20\u00b0C. Cell pellets
corresponding to 100 ml of cell culture were lysed by thawing on ice and sonication in buffer A (50
mM HEPES pH 7.5, 250 mM NaCl, 0.5 mM TCEP, 10 mM imidazole) supplemented with protease
inhibitors (Roche). Insoluble material was removed by high-speed centrifugation at 40,000 g for
a period of 1 hour at 4\u00b0C. The resulting supernatant was passed through a 5 \u03bcm filter and then
applied to a batch/gravity column containing 1 ml of TALON resin (TaKaRa Bio). The resin was
washed with 5 column volumes of buffer A to remove unbound material, before the addition of
buffer B to elute the retained protein (50 mM HEPES pH 7.5, 250 mM NaCl, 0.5 mM TCEP, 300
mM imidazole).
Fractions containing the Trx-Pol32 RBM fusion proteins were identified by SDS-PAGE,
pooled, and then applied to a Superdex S75 16 600 size exclusion chromatography column
(Cytiva) pre-equilibrated in buffer C (20 mM HEPES pH 7.5, 250 mM NaCl, 0.5 mM TCEP).
Fractions containing the recombinant protein were again confirmed by SDS-PAGE, pooled and
then concentrated to ~2.5 mg/ml by use of centrifugal concentrators with a 5 kDa molecular weight
cut-off (Sartorius). The purified protein was flash-frozen in aliquots and stored at -80\u00b0C until
required.
Purified synthetic peptides (Supplementary Table 2) were purchased from Peptide Protein
Research Ltd, Bishops Waltham, UK. All peptides used for fluorescence polarisation are labelled
at their N-terminus with Fluorescein followed by a glycine-tyrosine-glycine linker sequence (GYG).

Fluorescence polarisation
Fluorescein-labelled peptides (100 nM) were incubated at room temperature with increasing
amounts of purified Rfa1 NTD in 20 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 0.5 mM TCEP in
a black 96-well polypropylene plate (VWR). Fluorescence polarisation was measured in a
CLARIOstar multimode plate reader (BMG Labtech GmbH). All data were fitted by non-linear
regression to a one-site specific binding model, using Prism (v. 10.4, GraphPad Software),
allowing calculation of the reported dissociation constants (KD). For the competition experiment,
increasing amounts of an unlabelled Mph1 peptide were included in binding reactions containing
7 \u03bcM of Rfa1 NTD and 100 nM of the fluorescently labelled Pol32 pThr257 peptide.

Mass Spectrometric Analysis
For mass spectrometry sample preparation following non-radioactive protein kinase assays,
frozen Trx-Pol32 RBM samples were thawed, reduced with 5 mM TCEP for 1.5 h, and alkylated
with 10 mM iodoacetamide in the absence of light. Alkylation was then quenched by addition of 5
mM DTT. The samples were desalted using PD SpinTrap G-25 columns (GE Healthcare) and
digested with 2 \u03bcg of Trypsin Gold (Promega) at 37\u00b0C. After desalting using C18 tip-columns
(Thermo Fisher), samples were concentrated using a vacuum-concentrator. Finally, the samples
were reconstituted in 40 \u03bcL of a solution of 0.5% acetonitrile and 1% formic acid and optionally
subjected to titanium dioxide columns (Thermo Fisher) for phospho-peptide enrichment. LC-
MS/MS analysis was undertaken using a Vanquish Neo UHPLC coupled to an Orbitrap Exploris
480 equipped with FAIMS Pro Duo interface (Thermo Fisher Scientific), following a data-
dependent MS2 experiment with internal EasyIC (RunStart) as lock mass. In summary, samples
were loaded onto a PepMap Neo column (5 \u03bcm C18, 300 \u03bcm x 5 mm, 100 \u00c5 pore size; Thermo
Fisher Scientific) at a flow rate of 20 \u03bcL/min using 0.1% formic acid in H2O and separated on an
analytical EASY-Spray PepMap Neo column (2 \u03bcm C18, 75 \u03bcm x 500 mm, 100 \u00c5 pore size,
Thermo Fisher Scientific) with a flow rate of 350 nl/min with a linear gradient of 1 to 35% solvent
B (80% (v/v) acetonitrile, 0.1% (v/v) formic acid) over 60 min. Ion source conditions were
maintained at 1.75 kV positive voltage, 275\u00b0C ion transfer tube temperature and standard
resolution with 3.8 l/min FAIMS carrier gas flow throughout the acquisition. Mass spectrometric
data was acquired using the following configuration: MS1Orbitrap resolution was set at 60,000
(m/z 200) from a mass range of 375 to 1200 amu using a normalized automatic gain control (AGC)
target of 300% and maximum injection time (maxIT) set to auto; MS2 Orbitrap resolution was set
at 15,000 (m/z 200) with first acquired mass starting at 120 amu, an isolation window of 2 amu,
and AGC set to standard with 40 msec maxIT. Fragmentation was carried out using higher-energy
collisional dissociation (HCD) with a normalized collision energy (NCE) of 30. For the FAIMS, two
compensation voltages (CV), 45 and 65 V with 2 and 1 sec cycle time, respectively, for each CV
were used with a RF lens of 50%. The following mass filters were also used: MIPS (peptide mode),
intensity threshold (8.0E3), charge state (2-6) and dynamic exclusion (30 sec) mass filters were
used. Data was analysed using Proteome Discoverer 3.0 (Thermo Fisher Scientific) with Sequest
HT and MS Amada 2.0 as the search engines. An in-house modified peptide sequences FASTA
file (based on our Pol32 RBM construct) was used to search against. For both Sequest HT and
Amada engine precursor and fragment mass limits were fixed at 5 ppm and 0.02 Da. Only the
phosphorylation for serine, threonine, and tyrosine residues was set as dynamic modifications.
The protein FDR and the peptide spectrum match were both set to 0.01.

Modelling with AlphaFold and molecular graphics
Molecular models were generated using the AlphaFold Server available online at
https://alphafoldserver.com, which currently implements version 3 of AlphaFold38. Molecular
graphics were generated using PyMOL 3.1 (The PyMOL Molecular Graphics System, Version 3.0
Schr\u00f6dinger, LLC).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "DNA double-strand breaks (DSBs), if not properly repaired, can lead to the loss of chromosomal integrity. DSBs can be repaired through non-homologous end joining (NHEJ), which re-ligates broken ends, or through homologous recombination (HR). HR repair occurs via two principal mechanisms: gene conversion (GC) and break-induced replication (BIR). BIR becomes necessary when only one end shares homology with a donor, such as at stalled or broken replication forks or at dysfunctional telomeres.\nNumerous studies have established that Pol32 is not required for gene conversion but is essential for BIR. While DNA synthesis can initiate in the absence of Pol32, it does not proceed efficiently during BIR. How Pol32 regulates BIR remains a central question. In this study, the authors show that the interaction between Pol32 and RPA is critical for BIR. The data also suggest that Pol32 interacts with Mph1 and Pif1 via RPA, leading to a very interesting idea that RPA serves as a critical platform for the orchestration of the BIR machinery. The significance of the proposed RPA-Pol32 interaction is not fully clear because RBM deletion did not have significant impact on Pol32-mediated BIR (Fig. 1D). This finding suggests that other critical domains within Pol32 may be responsible for regulating the interactions between Pol32, RPA, Mph1 and Pif1 (Fig. 5).\nSpecific Points:\n1. In addition to the Pol32-RBM-Rpa1 NTD complex (Figures 2 and 4), Alphafold structural predictions of the entire PolD-RPA complex would assist in constructing a more comprehensive model (Figure 5).\n2. Does the assay (Figure 1D) fully exclude the possibility of GC between the two chromosomes? For instance, did the authors confirm the presence of two copies of LEU2 after DSB induction?\n3. The efficiency of DSB induction and cell survival following HO expression in each experiment should be shown. Curiously, the assay becomes more reproducible as cell survival decreases (Figures 1E, 2D, 3B).\n4. The explanation of the Pol32-CTD (Figure 2C) is missing. Pol32-CTD expression is significantly low (Fig. 2C) although any other C-terminal deletion mutants were expressed well (Fig. 1C). What region does the Pol32 CTD mutation contain?\n5. Ddc2, Mph1, and Pif1 interact with Rfa1 via the same interface (Figure 4), suggesting that these proteins compete with Pol32. Does the deletion of DDC2, MPH1, or PIF1 increase the Pol32-Rfa1 interaction (Figure 2C)? The authors should provide a better explanation of how the interplay between Pol32, MPH1, and PIF1 drives BIR. This would help clarify the role of Pol32 in DNA synthesis during BIR.\n6. The authors show that Rim11 is required for BIR. Is Rim11 specifically necessary for BIR but not GC? The phosphorylation of Thr-257 by Rim11 needs to be validated through both in vitro and in vivo experiments. Because Pol32-T257D mutations were tested in all the genetic assays (Fig. 3 and Fig. 4B), T257D peptides should be also evaluated in Fig. 4A.\n7. Pif1 appears to interact with the N-terminus of Rfa1 similar to Ddc2 and Mph1 (Fig. 4E). Pif1 peptides would be tested in Fig. 4A.", "reviewer_2": "This work presents interesting data suggesting a dynamic role of RPA in BIR, where RPA interactions with Polδ, Mph1, and Pif1 dictate the stability and extension of the recombination D-loop during BIR. The authors identify residues within the Pol32 component of Polδ that are responsible for interaction with RPA. They also investigate the role of serine/threonine phosphorylation within the same region of Pol32 where it interacts with RPA. While phosphorylation sites were previously described, their role remains unknown. The authors show that Pol32 phosphorylation stimulates the Polδ-RPA interaction and enhances BIR efficiency.\nOne of the strongest aspects of this work is the identification of Rim11 kinase as a likely regulator of Polδ phosphorylation, with rim11Δ mutants exhibiting the same low BIR phenotype as Pol32 phosphorylation mutants. However, somewhat unexpectedly, the pol32 mutant that abolishes interaction with RPA does not, by itself, show any deficiency in BIR. Finally, the authors also identify the C-terminal part of Mph1 as interacting with RPA and regulating BIR, likely by influencing D-loop stability. They propose an interesting model in which Rim11-mediated phosphorylation shifts Rfa1 interaction from Mph1 (D-loop unwinding) toward Polδ (D-loop extension), thereby stimulating BIR.\nThis is an interesting study that provides new mechanistic insights into BIR regulation.\nConcerns\nThe analysis of phenotypes is relatively weak, as it relies on a single BIR assay that is highly inefficient, even in wild-type cells (only ~20% complete BIR). The differences between wild-type and mutants are within a ±5% range for some mutants. Additionally, several interesting predictions can be drawn from the proposed model that should be tested in vivo. A few additional experiments would significantly strengthen this work:\n1. Test BIR using another assay. The allelic BIR assay, which is commonly used, would be ideal. Decreased D-loop extension often leads to specific phenotypes, such as half-crossovers, which are expected in rim11Δ mutants.\n2. Measure BIR kinetics in an efficient BIR assay. It would be informative to examine BIR kinetics in mutants such as pol32_RBM, rim11Δ, and pol32 phosphorylation mutants. In particular, pol32_RBM might exhibit a BIR delay.\n3. Examine crossover frequency in the Mph1 C-terminal mutant. The mph1ΔC (1-954) mutant shows reduced interaction with RPA and stimulates BIR, likely due to loss of its effect on D-loop unwinding. A key prediction is that this mutant should exhibit a higher level of crossover outcomes, which should be tested.\n4. Test the genetic interaction between Rim11 and Mph1. If Rim11-mediated phosphorylation of Pol32 enhances Polδ-RPA interaction and reduces the negative impact of Mph1-RPA on BIR, then rim11Δ mph1-954 double mutants should not exhibit any defects in BIR. Testing BIR efficiency in this double mutant would directly test the authors' model.\nMinor Comments:\nRim11 localization and function: Rim11 is typically localized in the cytoplasm. Is it known to phosphorylate any other nuclear proteins? Is there evidence that Rim11 directly phosphorylates Pol32?\nClarification in Lines 39-41: The work first demonstrated that BIR is limited in the repair of replication fork breakage—PMID: 26273056.", "reviewer_3": "In the manuscript by Jones and co-workers, the authors report the identification of a novel RPA binding motif (RBM) in the Pol32 subunit of the DNA polymerase Delta using the budding yeast as a model organism. Normally, the Pol32 recruitment to DNA is controlled through its interaction with PCNA via a PCNA-interacting peptide (PIP) located at the C-terminus of Pol32. The importance of the identified RBM in vivo, based on DNA repair by BIR, could only be revealed if the Pol32-PCNA interaction is eliminated from the system by the Pol32 C-terminal deletion involving the PIP, as shown by the experiments in figure 2D. The RPA-Pol31-Pol32 in vivo interaction is detected by protein CoIPs (Figure 2C).\nThe in vitro binding experiments and Alpha-fold modelling support the idea that phosphorylation of some S/T residues within the RBM motif strengthens the RPA-Pol32 interaction. The phosphor-site mutations have an expected effect on BIR in the background where the Pol32-PCNA interaction is lost, but behave in a confusing way in full length Pol32. The authors then claim that Rim11 is the kinase responsible for the phosphorylation of the RBM in Pol32.\nFinally, a structural parallel is drawn to the RBMs from Pif1 and Mph1, two other proteins known to be involved in BIR and carrying RBMs.\nI have two concerns about this manuscript which I would like to discuss separately:\n1. the suitability for publishing it in Nature Communications;\n2. the experimental part.\nAs presented, the focus of the manuscript is on identifying a novel RBM in Pol32. Based on the fact that this is just another RBM added to the several previously reported in different organisms, including some of those in budding yeast, the novelty of this manuscript doesn’t advance the field enough to grant a publication in NC, particularly since the biological significance of this RBM in Pol32 is unclear and its potential regulation through phosphorylation is not studied well enough. Based on the experiments in this manuscript, the RBM plays a secondary role to the PIP in the Pol32 recruitment in BIR: with the Pol32 PIP being functional, the RBM has no effect on the efficiency of BIR. The RBM may play a more important role in some other genome stability aspect independent of BIR, but it is not presented in this work and, therefore, it remains unclear how important it is.\nWith this said, I could see how the focus of the manuscript could be shifted to a more of a big-picture question, to stress the novelty of identifying extended RBMs where phosphorylation of the residues adjacent to the hydrophobic cores plays a role in genome stability via stimulating the recruitment of the repair proteins to DNA. This would be a major revision and require a lot of additional experiments, such as analysing multiple RBMs, the ones in Pif1 and Mph1 at the very least, demonstrating the phosphorylation dependence on the DNA damage response (or other trigger) in vivo, different forms of peptide binding to RPA similar to those for Pol32 in figure 4A, plus addressing the physiological role of RBM phosphorylation in BIR through analysing the phosphosite mutants in the genetic BIR assays. These experiments would then justify the model proposed by the authors in Figure 5 and might make the story conceptually novel enough for NC to consider. As is, there is very little experimental data to support this model: the phosphorylation of RBM in Pol32 is not important for BIR in vivo; the phosphorylation of RBMs in Pif1 and Mph1 hasn’t been addressed experimentally. Also, it is not known what triggers the RBM phosphorylation – cell cycle, DNA damage signalling, etc. Therefore, the model in figure 5 is based on computer modelling and imagination, but not supported by sufficient experimental data.\nThe experimental part:\nFigure 1F. The authors use HU to assay different POL32 alleles. The rest of the paper is focused on BIR, i.e. DSB repair involving activation of the G2/M checkpoint rather than an intra-S phase checkpoint triggered by HU. It might be informative to add a spot test using Phleomycin or a similar drug inducing DSBs rather than stalled forks.\nFigure 2C. My understanding is that the C-terminal truncation form of Pol32 used in this experiment doesn’t have either RBM or PIP. It would be much more informative to use an RBM-only deletion mutant instead and present a similar experiment later in the paper when the RBM is identified. A separate point – if the authors suggest that the RBM might be phosphorylated to facilitate the repair of DNA damage (Figure 5), then it would make sense to do a pull-down from the cells with and without DNA damage induced.\nFigure 3A. The BIR for T257A is very confusing. The allele acts as dominant negative and the authors suggest a somewhat speculative explanation in the discussion. My suggestion would be to look for another RBM in Pol-delta as the mutated one in Pol32 might be blocking the access to an alternative RBM elsewhere in Pol-delta. In Figure 2C, the pulldown of Pol31 brings some RPA even in the absence of the C-terminus of Pol32 (last lane in the blot) suggesting another interaction point between Pol-delta and RPA.\nFigure 3C-D. It is very puzzling that the authors didn’t consider Chk2 kinases (Rad53 and Dun1) as the candidates for the Pol32 phosphorylation, particularly in the light of their model in Figure 5 and the fact that BIR strongly depends on Rad9 involved in Rad53 activation. The genetics presented in panels C and D is not easy to interpret conclusively, mainly because the T257A mutation in the full length Pol32 context has a hard-to-explain phenotype (Figure 3A, see above). However, T257A behaves much more predictably in Pol32(1-333). This is why the genetics should be done in this background by comparing BIR in Pol32(1-333), Pol32(1-333) Rim11-deletion, Pol32(1-333)-T257A Rim11-deletion, Pol32(1-333) T257D Rim11-deletion. If the genetic experiments still hold Rim11 relevant (it will only work if Rim11 has Pol32 as the only target in BIR), a physical interaction should be addressed between Pol32 (or Pol-delta) and Rim11. As presented, the evidence for the regulation of Pol32 by Rim11 is not convincing and unless more data are added, this part should be excluded from the manuscript.\nFigure 4C. The C-terminal deletion (1-953) truncates way too much to make a conclusion about the relevance of the newly identified RBM in Mph1 for BIR. The deleted sequence may contain another functional motif between aa 953 and 982. Even the Mph1 peptide used in the RPA binding assays (Figure 4D) starts from aa971, suggesting that there was no need for such an extended deletion. Therefore, a much shorter truncation (1-982) or a cluster of aa substitution in RBM should be used in the genetic experiments to address the importance of RBM in Mph1."}, "author_response": "We thank the referees for their assessments and suggestions. We were pleased they feel “this is an interesting study that provides new mechanistic insights into BIR regulation” (referee 2) by the “identification of a novel RPA binding motif (RBM) in the Pol32 subunit of the DNA polymerase Delta” (referee 3), whose role “in regulating BIR remains a central question” (referee 1). Their valuable comments have formed the basis for a major experimental revision including 11 new main figure panels and 11 new supplementary figure panels. We feel our additional work has significantly strengthened the study and improved our model for phospho-activated Pol32-RPA interactions during BIR-mediated DNA double-strand break repair. Below, please find our point-by-point response to the comments provided, a description of additional experiments, and details of changes made to the figures and the revised MS text (highlighted green in the MS text file).\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nDNA double-strand breaks (DSBs), if not properly repaired, can lead to the loss of chromosomal integrity. DSBs can be repaired through non-homologous end joining (NHEJ), which re-ligates broken ends, or through homologous recombination (HR). HR repair occurs via two principal mechanisms: gene conversion (GC) and break-induced replication (BIR). BIR becomes necessary when only one end shares homology with a donor, such as at stalled or broken replication forks or at dysfunctional telomeres.\nNumerous studies have established that Pol32 is not required for gene conversion but is essential for BIR. While DNA synthesis can initiate in the absence of Pol32, it does not proceed efficiently during BIR. How Pol32 regulates BIR remains a central question. In this study, the authors show that the interaction between Pol32 and RPA is critical for BIR. The data also suggest that Pol32 interacts with Mph1 and Pif1 via RPA, leading to a very interesting idea that RPA serves as a critical platform for the orchestration of the BIR machinery. The significance of the proposed RPA-Pol32 interaction is not fully clear because RBM deletion did not have significant impact on Pol32-mediated BIR (Fig. 1D). This finding suggests that other critical domains within Pol32 may be responsible for regulating the interactions between Pol32, RPA, Mph1 and Pif1 (Fig. 5).\nAuthors’ response: We agree with Referee 1 that the question of how Pol32 regulates BIR remains an important question in the field. In our study we discover a hitherto unknown RPA binding module (RBM) in Pol32 which mediates a novel physical interaction between Pol d and RPA. What is particularly intriguing is that the Pol32 RBM provides the first example of a phospho-activated RBM whose affinity to the Rfa1 binding groove is increased more than 150-fold by phosphorylation at Thr256 and Thr257. While additional protein interactions between Pol32 and PCNA or Pol a contribute to BIR efficiency, it appears that phospho-activation is unique to the Pol32-RPA interaction. We therefore propose that the significance of this new Pol32 RBM lies in its potential to act as dynamic regulator of BIR. Consistently, we demonstrate that phospho-mimetic amino acid substitutions T256D/T257D boost BIR efficiency in vivo. The corresponding phospho-null substitutions negatively impact BIR efficiency, which becomes apparent in the absence of the Pol32 PIP box (new Fig. 3G); moreover, new kinetic analyses of BIR show that mutations within the RBM which lower the affinity for RPA result in slower repair kinetics and a severe repair defect in the absence of the Pol32 PIP-box (new Fig. 2E).\nEqually important, we define a new RBM in the BIR antagonist Mph1 and show that it interacts with the same binding groove in Rfa1 as those in Pol32 and Pif1. We demonstrate that the Pif1 and Mph1 RBMs bind RPA with high affinity in the non-phosphorylated state (new Fig. 4B and Fig. 4D), which can be explained by the presence of negatively charged amino acid residues at positions equivalent to the Thr256/257 double phosphorylation site in the Pol32 RBM.\nThese findings support a mechanism by which cells can manipulate the interplay of the core enzymes involved in BIR at RPA-bound D-loops, thereby allowing dynamic control over D-loop stability and BIR efficiency levels. This model does not require invoking additional, unidentified functional domains within Pol32 for consistency, and adds an important dimension to our understanding of how BIR outcomes are regulated. Beyond BIR, the phospho-regulation of the Pol32 RBM serves as a paradigm for the malleability of RPA interactions in the regulation of DNA metabolic processes, opening the door to future studies on phospho-regulated RPA interactions.\nSpecific Points:\n1. In addition to the Pol32-RBM-Rpa1 NTD complex (Figures 2 and 4), Alphafold structural predictions of the entire PolD-RPA complex would assist in constructing a more comprehensive model (Figure 5).\nAuthors’ response: We thank Referee 1 for this suggestion and have now performed additional AlphaFold3 (Abramson J et al., Nature 630:493–500 (2024). doi: 10.1038/s41586-024-07487-w) analyses of the Pol32-RPA interaction in the context of the yeast Pol d holocomplex engaged with PCNA and a primer-template junction to further inform the model presented in revised Fig. 5. Specifically, we have modelled full-length Pol32, bis-phosphorylated at Thr256 and Thr257 (please also refer to point 6 below) as part of the Pol d-PCNA-DNA complex and included the N-terminal domain of Rfa1 (Rfa1 NTD), which is attached to the remainder of the RPA trimer by a long linker. In brief, the modelling revealed several features not captured by an existing Pol d-PCNA-DNA cryo-EM structure (PDB entry 7KC0; Zheng F et al, Proc Natl Acad Sci U S A 117:30344-30353 (2020). doi: 10.1073/pnas.20176371172020), which does not resolve the unstructured Pol32 C-terminal domain (CTD). Thus, the AlphaFold3 models consistently show multiple interactions of the Pol32 CTD within the Pol d-PCNA-DNA-Rfa1-NTD assembly: (1) In all predicted models, an as-yet undescribed helix-turn-helix motif at Pol32 amino acid residues 164-218 (which we now refer to as ‘Hook’) binds a monomer-monomer junction of the PCNA trimer (i.e., not to the interdomain connector loop (IDCL) which is the target of canonical PIP-box interactions). This interaction is reminiscent of a previously described Rev1-PCNA interaction (Sharma NM et al. (2011) J Biol Chem 286:33557-66. doi: 10.1074/jbc.M110.206680). In contrast to the Pol32 PIP-box, the Pol32 ‘Hook’ is not sufficient to support apparent PCNA interactions by yeast-two-hybrid assay or BIR efficiency. Nonetheless, this new feature is noteworthy as its similarity to Rev1 suggests that it provides an additional PCNA contact point for Pol d. (2) In 5/5 models, the Pol32 PIP-box is engaged with one of three available IDCLs within the PCNA homotrimer, indicating sufficient Pol32 CTD flexibility to ensure that the Pol32 PIP-box can engage PCNA when Pol d is bound at the D-loop primer junction. (3) The phosphorylated Pol32-RBM is consistently engaged with the Rfa1 NTD across models, and the flexibility and length of the linker between the Rfa1 NTD and subsequent RPA domains indicates that Pol32 can interact with RPA which is bound to the displaced ssDNA strand at a D-loop while Pol d-PCNA sits at the primer junction during BIR.\nThese new insights provide an important refinement to our model (now presented as revised Fig. 5D), showing the potential of Pol32 to recruit and stabilize Pol d at the primer-template junction DNA through two-pronged PCNA and RPA interactions, thereby promoting BIR. The above is described in detail in the revised version of the MS in a dedicated new Results section entitled “A model for the actions of Pol32 in BIR” (page 13, line 343-page 14, line 379) and illustrated in new Fig. 5, panels A-C (and new Supplementary Fig. S9).\n2. Does the assay (Figure 1D) fully exclude the possibility of GC between the two chromosomes? For instance, did the authors confirm the presence of two copies of LEU2 after DSB induction?\nAuthors’ response: Yes, the data shown in Fig. 1D dos exclude GC. The BIR assay does report on (Pol32-independent) GC, and it has been determined that the GC rate after HO induction in JRL-derived strains is ~2% (Lydeard JR et al., Nature 448:820-3 (2007). doi: 10.1038/nature06047). GC-dependent repair of the HO endonuclease-induced DSB on chromosome V in JRL-derived strains (JRL092 and YRL629-YRL648, and new strains YRL661-YRL666, YRL679, YRL680 in the revised MS) results in the retention of the HPH gene on chromosome V. In good agreement with GC rates reported by Jim Haber’s lab, we routinely find 2-4% of DSB repair survivors in JRL-derived strains have retained HPH (which are consequently resistant to hygromycin B selection), regardless of any specific mutation we have tested. This information is now included in the Methods section of the revised version of the MS as detailed in our reply to point 3 below.\n3. The efficiency of DSB induction and cell survival following HO expression in each experiment should be shown. Curiously, the assay becomes more reproducible as cell survival decreases (Figures 1E, 2D, 3B).\nAuthors’ response: The HO endonuclease is a yeast-endogenous enzyme and its efficiency in reliably generating a site-specific DSB across an entire yeast cell population if expressed from a GAL promotor is the basis for widely used HO-based BIR reporter assays. Thus, loss of Rad51, which is required for BIR and GC renders JRL-derived strains virtually inviable upon HO induction by galactose in the culture medium (Lydeard JR et al., Nature 448:820-3 (2007). doi: 10.1038/nature06047; Lydeard JR et al., Genes Dev. 24:1133-44. (2010) doi: 10.1101/gad.1922610). Ineqicient cutting is not considered a contributing factor to the distribution of repair eqiciency data obtained with this assay. To demonstrate the effectiveness of the GAL-induced HO endonuclease in our hands, we have now included in the revised version of the MS a PCR-based validation of DSB formation (new Supplementary Fig. S1A), as well as of the de novo generation of a contiguous CAN1 gene through BIR-dependent DSB repair in HO cut survivors (new Supplementary Fig. S1B), supplementing Fig. 1E. We routinely find that a PCR product spanning the HOcs locus can no longer be amplified when the HO endonuclease is induced on YPGAL medium, indicating eqicient chromosome V breakage, which in line with a survival rate after HO induction that tends towards 0% in strains lacking POL32 (residual survival of 2-4% occurs by GC, generating hygromycin B-resistant colonies which are not included when reporting BIR eqiciency). To clarify, the revised MS text has been amended on page 4, lines 92-98 to read:\n“The impact of these mutations on BIR was determined using a reporter strain23 for BIR eIiciency at a DSB on chromosome V with the only available repair template located at an ectopic site on chromosome XI (Fig. 1D). The DSB is eIectively introduced across the cell population upon galactose-induced expression of the HO endonuclease23 (Supplementary Fig. S1A). BIR-mediated DSB repair can be genetically tracked as it restores a CAN1 marker at the cut site whilst leading to the loss of a strategically placed HPH marker (Fig. 1D and Supplementary Fig. S1B).”\nAnd additional information has been added to the Methods section entitled “BIR e@iciency measurements” in the revised version of the MS on page 18, lines 470-484:\n“To measure ectopic BIR (JRL092-derived strains), logarithmically growing cells cultured in YPRAFF medium were plated in appropriate dilutions onto YPD or YPGAL medium and grown into colonies. Cell viability as a measure of DSB repair eIiciency after HO endonuclease induction was derived by dividing the number of colony-forming units (CFU) on YPGAL by that on YPD. For JRL-derived strains, HO cut eIiciency was routinely tested by PCR, and a PCR product spanning the HOcs locus was no longer obtained after exposing cells to galactose for 1 h (for an example, see Supplementary Fig. S1A). Colonies on YPGAL were always replica plated onto YPD medium supplemented or not with 200 µg/ml hygromycin B. This showed that 2-4% of DSB repair events led to retention of the HPH resistance marker, in good agreement with previously determined23 rates of BIR-independent DSB repair by gene conversion in JRL-derived strains. This fraction was subtracted from the value for DSB repair eIiciency to calculate BIR eIiciency for a given strain. In addition, BIR survivors were routinely validated using PCR on random colonies to show the presence of a new, contiguous CAN1 marker at the former HOcs locus (see Supplementary Fig. S1B).”\n4. The explanation of the Pol32-CTD (Figure 2C) is missing. Pol32-CTD expression is significantly low (Fig. 2C) although any other C-terminal deletion mutants were expressed well (Fig. 1C). What region does the Pol32 CTD mutation contain?\nAuthors’ response: The Pol32DCTD mutant is nearly identical to Pol32_1-249. However, following the suggestion by Referee 3, we have replaced the Pol32DCTD mutant in IP experiments (former Fig. 2C) with the more refined Pol32DRBM mutant, which harbours an internal deletion of the newly identified RBM only rather than a deletion of the entire CTD (new Fig. 2B in the revised version of the MS). The Pol32DRBM mutant shows much more similar protein levels to wild-type Pol32 in native protein extracts used for IP experiments as compared to Pol32DCTD. Importantly, after IP, we observe very similar amounts of recovered Pol d holoenzyme for complexes containing full-length Pol32 or Pol32DRBM, while the latter (like complexes containing Pol32DCTD in the previous version of the MS) show a severe reduction of co-recovered RPA, validating more elegantly that the novel Pol d-RPA protein-protein interaction is mediated by the Pol32 RBM. This is referred to in the revised version of the MS on page 6, line 162-page 7, line 166:\n“However, an internal deletion of Pol32 aa 247 to 258 (hereafter referred to as DRBM) diminished Pol d-RPA interactions (Fig. 2B). These data are therefore consistent with the newly identified acidic-hydrophobic motif within Pol32 functioning as a bona fide RBM and serve to confirm the novel Pol d-RPA interaction.”\n5. Ddc2, Mph1, and Pif1 interact with Rfa1 via the same interface (Figure 4), suggesting that these proteins compete with Pol32. Does the deletion of DDC2, MPH1, or PIF1 increase the Pol32-Rfa1 interaction (Figure 2C)? The authors should provide a better explanation of how the interplay between Pol32, MPH1, and PIF1 drives BIR. This would help clarify the role of Pol32 in DNA synthesis during BIR.\nAuthors’ response: We have previously shown that Ddc2-RPA interactions (Yates LA et al., Proc Natl Acad Sci U S A 120:e2300150120 (2023) doi: 10.1073/pnas.2300150120.) support DNA damage checkpoint signalling upstream of Rad51-mediated strand invasion and do not expect an interplay between Ddc2 and Pol32 at D-loop primer junctions. However, we have performed the suggested experiment by deleting MPH1 or PIF1 in strains harbouring endogenously TAP-tagged Pol31 and analysing TAP-IPs for RPA recovery. While we detected slight increases in RPA recovery in the deletion strains (Rfa1/Pol31-TAP signal ratio for control = 1; 1.23 and 1.19 for the pif1D and mph1D strains, respectively), we do not think that these minor effects provide relevant information on the interplay of Pol32, Mph1, and Pif1. This is because RPA is estimated to be present in cells at 5-10 x higher concentrations than Pol32, Mph1, or Pif1 (https://www.yeastgenome.org/). It is therefore unlikely that the saturation of immunoprecipitated Pol d complex with RPA would be significantly changed in pif1D and mph1D cells regardless of the ability of Mph1 and Pif1 to occupy the same protein binding site on Rfa1 as Pol32. As illustrated in the model in revised Fig. 5D, similar considerations apply to the in vivo situation at BIR intermediates with a multitude of RPA molecules bound to associated ssDNA regions. Here, RPA provides a platform for the engagement of Pol32, Mph1, and Pif1 through their respective RBMs which bind to the basic-hydrophobic groove of Rfa1 subunits, thereby increasing their local concentration and effecting BIR outcomes. How significant a direct competition for the Rfa1 basic-hydrophobic groove on the same Rfa1 molecule may be, remains an open question for future study; answering this question will likely require fully reconstituted BIR single-molecule studies, which goes beyond the scope of our current study.\nHowever, given that the 3'-DNA junction at a D-loop can only be engaged either by Pol d (promoting further DNA synthesis) or Mph1 (unwinding and D-loop disassembly), it is conceptually straightforward that direct competition between Pol32 and Mph1 for RPA can be relevant locally and affect BIR efficiency (of note, Mph1 does not contain a PIP-box and can therefore not rely on PCNA for recruitment to the primer junction). We articulate this point on page 15 (lines 405-416) in the revised version of the MS:\n“Mph1 targets extended D-loops24,49 and dissociates the nascent strand47 to restrict BIR efficiency48,60. We find that these actions of Mph1 depend on a newly defined RBM at the Mph1 C-terminus. The Mph1 RBM binds RPA more avidly than the non-phosphorylated Pol32 RBM, but with similar affinity to the phosphorylated form. It is therefore tempting to speculate that Pol32 RBM phosphorylation may help Pol d to locally resist the actions of Mph1 at the D-loop primer junction. Thus, Pol32 RBM phosphorylation may promote BIR by stabilizing Pol d at the primer junction and mitigating D-loop collapse. As a consequence, the risk of chromosome aberrations5,6 arising from ectopic recombination upon premature release of the nascent strand and invasion at a non-contiguous site with sequence homology61-64 could be reduced. Conversely, Pol32 RBM de-phosphorylation may favour D-loop disassembly, allowing cells to terminate BIR and limit potentially mutagenic8 BIR-dependent DNA synthesis.”\nLike Pol32, Pif1 is a BIR agonist, and unlike Mph1, Pif1 does not require competitive access to the primer junction engaged by Pol d. To drive D-loop progression, Pif1 is thought to contact the displaced ssDNA strand as shown in our model in revised Fig. 5D. As suggested by the Makovets lab, this is likely to be aided by Pif1 RBM-RPA interactions (Kotenko O & Makovets S. EMBO Rep 25:1734-1751 (2024) doi:10.1038/s44319-024-00114-9). Consistently, abrogation of the Pif1 RBM brings down BIR efficiency (new Fig. 4C in the revised version of the MS). Coordination of Pif1 and PCNA-bound Pol d during BIR is mediated by PCNA, to which Pif1 binds with its own PIP-box (Buzovetsky O et al. Cell Rep 21:1707-1714 (2017) doi: 10.1016/j.celrep.2017.10.079). We suggest that “our model is consistent with the independent contributions which we have observed the Pol32 RBM and PIP-box make to BIR efficiency. We propose that multi-point interactions of Pol32 and Pif1 (refs. 20,59) with PCNA and RPA-bound recombination intermediates ensure the proper recruitment and retention of Pol d and the accessory D-loop helicase Pif1 at BIR intermediates, allowing optimal BIR reactions to take place (Fig. 5D).” (page 14, lines 374-379 in the revised version of the MS).\n6. The authors show that Rim11 is required for BIR. Is Rim11 specifically necessary for BIR but not GC? The phosphorylation of Thr-257 by Rim11 needs to be validated through both in vitro and in vivo experiments. Because Pol32-T257D mutations were tested in all the genetic assays (Fig. 3 and Fig. 4B), T257D peptides should be also evaluated in Fig. 4A.\nAuthors’ response: We thank Referee 1 for suggesting further investigation of Pol32 RBM phosphorylation by in vitro kinase assays. We have now tested Rim11 and were surprised to find that despite the GSK3 family kinase consensus sequence within the Pol32 RBM, Pol32 is a poor phosphorylation substrate (new Supplementary Fig. S4). This suggested that (1) Rim11 impacts BIR through a phosphorylation target other than Pol32, and that (2) Pol32 RBM phosphorylation in vivo (refs. 40-44 in the revised version of the MS) is mediated by a set of implicated kinases which remain to be tested in vitro.\nRegarding point (1), we considered the characterized phosphorylation targets of Rim11, which include the meiosis-specific protein Ime1 (Rubin-Bejerano I et al., Mol Cell Biol 24:6967-79 (2004). doi: 10.1128/MCB.24.16.6967-6979.2004), the cytosolic Pah1 phosphatidate phosphatase (Khondker S et al., J Biol Chem 298:102221 (2022). doi: 10.1016/j.jbc.2022.102221), and the Ume6 subunit of the Ume6-Rpd3 histone deacetylase complex (Malathi K et al., Mol Cell Biol 17:7230-6 (1997). doi: 10.1128/MCB.17.12.7230). Interestingly, Pif1 is a deacetylation target of Ume6-Rpd3, providing a potential link to BIR (Ononye OE et al., J Biol Chem 295:15482-15497 (2020). doi: 10.1074/jbc.RA120.015164). Indeed, we found that deletion of UME6 reduces BIR efficiency similarly to a deletion of RIM11 (Figure A herein), providing a potential explanation as to why Rim11 loss negatively affects BIR efficiency. While the role of Rim11 in BIR warrants further investigation, for the purposes of the current study the new data clearly indicate that Rim11 is not the kinase responsible for the phospho-regulation of the Pol32 RBM.\nFig. A: Left, Rim11 has been implicated in the phosphorylation of a limited number of potential (light blue) and verified (dark blue/yellow) targets. Among them, Ume6-Rpd3 targets Pif1 whose acetylation status impacts Pif1 activity. This connects the Rim11 kinase with Pif1-dependent BIR, providing a potential explanation for the observation that loss of RIM11 results in a BIR efficiency defect. Right, BIR efficiency measurements for the indicated JRL-derived strains revealing a BIR defect upon loss of UME6 that is similar to the one observed upon RIM11 loss.\n(2) To address which kinase might directly phosphorylate Pol32 RBM, we first considered the kinases that have been linked to Pol32 phosphorylation at residues within the RBM in vivo. Phospho-proteomic studies consistently show that the Pol32 RBM can be phosphorylated at Thr257, and also at Thr256 and Ser264 by multiple potential kinases (refs. 40-44 in the revised version of the MS): (i) Dbf4-dependent kinase (DDK) Cdc7, (ii) cyclin-dependent kinase (CDK) Cdc28, and (iii) Polo-like kinase (PLK) Cdc5. Given the acidic-hydrophobic amino acid sequence surrounding Thr257, we also considered the acidophilic casein kinase CKII, although there is currently no available in vivo evidence to support CKII-dependent Pol32 RBM phosphorylation. We used Pol32 RBM fragments spanning amino acid residues 248 to 264 fused to thioredoxin in in vitro kinase assays with TAP-tagged versions of Cdc7, Cdc5, Cdc28, and CKII. Affinity-captured Cdc7, Cdc5, and CKII were able to phosphorylate the RBM fragment, but not Cdc28 (nor Rim11) (new Supplementary Fig. 4A, B). Phosphorylation levels dropped when Cdc7, Cdc5, and CKII were offered a version of the RBM fragment harbouring amino acid substitutions T256A/T257A (new Supplementary Fig. 4B). Next, we performed mass spectrometry phosphorylation analysis of the wild-type Pol32 RBM peptide to reveal that while all affinity-purified kinases exerted detectable kinase activity on the thioredoxin fusion, the Pol32 RBM was only phosphorylated by Cdc7, Cdc5, and CKII at Thr256, Thr257, and Ser264 (new Fig. 3A, B and new Supplementary Fig. 4C). The strongest activity was exhibited by the acidophilic kinases CKII and Cdc7. Interestingly, Cdc7 has been previously reported to promote BIR efficiency (Lyderad JR et al., Genes Dev 24:1133-44 (2010). doi: 10.1101/gad.1922610). These extensive new data confirm the DDK and PLK-dependent phosphorylation of the Pol32 RBM observed in vivo and suggest that Cdc5, Cdc7, and possibly additional acidophilic kinases such as CKII, contribute to establishing the ‘phospho-code’ on Pol32. The experiments are described in an entirely new section in Results (please see section “The Pol32 RBM can be phosphorylated by multiple protein kinases.”; page 7, line 190-page 8, line 219) and in the Discussion on page 16 (line 417-page 17, line 436), as follows:\n“Regulation of the Pol32 RBM phosphorylation status may involve multiple kinases. Ddc2 RBM Ser 10 and Ser11 phosphorylation is driven by the constitutively active acidophilic kinase CKII and its regulation is likely to involve the actions of phosphatases.37 Consistent with the sequence similarity between the Ddc2 and Pol32 RBMs, we find that CKII can also phosphorylate the Pol32 RBM in vitro and may therefore contribute to the phospho-regulation of Pol32. Several other kinases have been implicated in Pol32 RBM phosphorylation in vivo. 40-44 We confirmed the ability of Cdc5 (PLK) and Cdc7 (DDK) to directly phosphorylate the Pol32 RBM in vitro. Phospho-proteomics suggest that Cdc7 phosphorylates serine and threonine residues with Asp/Glu/pSer/pThr at the plus 1 position44, and bis-phosphorylation at Thr256/257 within Pol32 conforms with this preference. Interestingly, Cdc7 showed the most robust activity towards the Pol32 RBM in vitro and has previously been reported46 to promote BIR in vivo. DDK is active from the start of S-phase through to mitosis, while PLK is activated at the G2/M transition. Both kinases act to align cellular processes with cell cycle stage and are known to co-regulate recombination-associated reactions, for example by coordinating the resolution of recombination intermediates with mitotic entry65. Their activity towards Pol32 would suggest that the Pol32 RBM becomes increasingly activated for high-affinity RPA binding as cells progress through S phase, which could promote effective BIR at broken replication forks or exogenous DNA breaks in S/G2 phase. However, the details of Pol32 phospho-status regulation, which may involve additional kinases as well as phosphatases, remain to be elucidated in future studies.”\n(3) To validate that Pol32 RBM phospho-mimetic mutations mediate an increase in affinity for the Rif1 NTD as expected from our experiments with the phosphorylated Pol32 RBM peptides, we have now performed an FP experiment with a Pol32 T256D T257D peptide. The threonine to aspartate substitutions significantly increase the affinity of the Pol32 RBM for Rfa1 NTD as compared to the non-phosphorylated wild-type peptide, improving the K from 22.9 µM to 2.93 µM. We agree with Referee 1 that this is a valuable control for the link between increased Pol32 RBM-RPA affinity and enhanced BIR efficiency. This new data is presented in new Supplementary Fig. S6C and referred to in the revised version of the MS on page 10, line 270-page 11, line 283:\n“Addressing the impact of enhanced Rfa1 NTD interactions mediated by Pol32 pRBM on BIR, we confirmed that phospho-mimetic amino changes Thr256Asp and Thr257Asp within the RBM increase the affinity for Rfa1 (Supplementary Fig. 6C). We then generated versions of the ectopic BIR reporter strain expressing Pol32 T256D T257D, which was present at concentrations comparable to wild-type Pol32 (Fig. 3E). Importantly, Pol32 T256D T257D boosted BIR efficiency as compared to the control strain, identifying a first Pol32 mutant that improves BIR (Fig. 3F). Like Pol32 DRBM and -RBM*, an endogenously expressed Pol32 T256A T257A phospho-null mutant was not associated with a BIR efficiency defect (Fig. 3F). However, as in the case of Pol32_1-333 RBM*, a strain expressing Pol32_1-333 T256A T257A showed a significant drop in BIR efficiency, suggesting that phosphorylation at Thr256/257 is required for BIR efficiency in the absence of the Pol32 PIP-box. Consistently, T256D/T257D continued to enhance BIR efficiency in the context of a Pol32 PIP-box truncation mutant (Fig. 3G). These data are in line with the notion that the phosphorylation of the Pol32 RBM in vivo40-44 can be deployed by cells to modulate BIR efficiency.”\n7. Pif1 appears to interact with the N-terminus of Rfa1 similar to Ddc2 and Mph1 (Fig. 4E). Pif1 peptides would be tested in Fig. 4A.\nAuthors’ response: We have now tested a Pif1 RBM peptide with Rfa1 NTD in a fluorescence resonance assay as shown in former Fig. 4A. The measured K is 0.32 µM, showing that the Pif1 RBM binds Rfa1 with high affinity more akin to the phosphorylated Pol32 RBM and much tighter than the non-phosphorylated form of the Pol32 RBM (new Fig. 4B in the revised version of the MS). This is consistent with our AlfoldFold modelling showing that Trp75 of the non-phosphorylated Pif1 RBM reaches into a well-defined hydrophobic ‘side-pocket’ on the surface of Rfa1 NTD, mirroring an interaction that makes important contributions to the strength of RBM-Rfa1 NTD interactions for various other RPA client proteins (Wu Y et al., Elife 12:e81639 (2023) doi: 10.7554/eLife.81639). Additional AlphaFold modelling shows that the ‘side-pocket’ interaction is not significantly altered by Pif1 RBM phosphorylation (new Supplementary Fig. S7E). Consistently, phosphorylation of Ser70 and Ser72 within the Pif1 RBM results in only a minor change in affinity for the Rif1 NTD with an apparent K of 0.15 µM (new Supplementary Fig. S8A in the revised version of the MS). In contrast, the Pol32 RBM requires phosphorylation to facilitate the conformational change that enables an equivalent ‘side-pocket’ interaction mediated by Leu262 for high-affinity interactions. The relevant revisions read (page 11, line 290-page12, line 310; page 13, lines 336-341):\n“While the Pif1 RBM has been defined as spanning aa 60 to 74 (ref. 20), Trp75 within Pif1 aligns well with a similar hydrophobic residue, Phe993, at the end of the putative RBM sequence in Mph1 (Fig. 4A). Interestingly, both Pif1 and Mph1 RBMs contain pre-existing negative charges (Asp71 and Asp990, respectively) at the position of the Thr256/257 double phosphorylation site within Pol32, suggesting that both RBMs might behave more similarly to the phosphorylated rather than the non-phosphorylated Pol32 RBM. Indeed, AlphaFold modelling predicts that the Pif1 and Mph1 RBMs bind Rfa1 with the same polarity as Pol32, placing three hydrophobic residues consistent with the f-D/E-x-f-f motif into the Rfa1 groove: Pif1 Leu66, -Leu68 and -Leu69; Mph1 Leu983, -Ile986 and -Leu987 (Fig. 4A and Supplementary Fig. S7A, B). Pif1 Trp75 and Mph1 Phe993 bind directly into the hydrophobic ‘side-pocket’ that is occupied by Leu262 in the phosphorylated forms of the Pol32 RBM (Supplementary Fig. S7C, D). Here, the additional phosphorylation of the Pif1 or Mph1 RBMs is not predicted to significantly impact the position of Pif1 Trp75 and Mph1 Phe993, which suggests that both proteins adopt the high-affinity Rfa1 binding confirmation independently of phosphorylation within their RBMs (Supplementary Fig. S7E, F). Consistently, we determined a K of 0.32 µM (95% CI, 0.25-0.52 µM) for a fluorescein-labelled peptide derived from Pif1 aa 59 to 79 with the Rfa1 NTD by FP (Fig. 4B). Thus, Pif1 tightly binds RPA and with similar affinity to Pol32 RBM phosphorylated at Thr256/257. Phosphorylation of Pif1 at Ser70 and Ser72 flanking Asp71 had a moderate impact on Rfa1 NTD binding affinity; improving the K to 0.15 µM (95% CI, 0.13-0.16 µM; Supplementary Fig. S8A).”\n“Overall, these findings show RBM-mediated RPA interactions of each BIR replisome component and BIR antagonist Mph1 impact homologous recombination-dependent DNA synthesis, highlighting a critical scaffolding function of RPA bound at recombination intermediates. Pif1 and Mph1 intrinsically bind RPA with high affinity, whereas phosphorylation is required to activate high-affinity RPA binding by the Pol32 RBM, providing a mechanism for the dynamic regulation of BIR efficiency (Fig. 4F).”\nReviewer #2 (Remarks to the Author):\nThis work presents interesting data suggesting a dynamic role of RPA in BIR, where RPA interactions with Polδ, Mph1, and Pif1 dictate the stability and extension of the recombination D-loop during BIR. The authors identify residues within the Pol32 component of Polδ that are responsible for interaction with RPA. They also investigate the role of serine/threonine phosphorylation within the same region of Pol32 where it interacts with RPA. While phosphorylation sites were previously described, their role remains unknown. The authors show that Pol32 phosphorylation stimulates the Polδ-RPA interaction and enhances BIR efficiency.\nOne of the strongest aspects of this work is the identification of Rim11 kinase as a likely regulator of Polδ phosphorylation, with rim11Δ mutants exhibiting the same low BIR phenotype as Pol32 phosphorylation mutants. However, somewhat unexpectedly, the pol32 mutant that abolishes interaction with RPA does not, by itself, show any deficiency in BIR. Finally, the authors also identify the C-terminal part of Mph1 as interacting with RPA and regulating BIR, likely by influencing D-loop stability. They propose an interesting model in which Rim11-mediated phosphorylation shifts Rfa1 interaction from Mph1 (D-loop unwinding) toward Polδ (D-loop extension), thereby stimulating BIR.\nThis is an interesting study that provides new mechanistic insights into BIR regulation.\nAuthors’ response: We thank Referee 2 for their positive assessment of our work. We are happy to report that the puzzling discrepancy between the Pol32 T257A phosphorylation mutant and Pol32DRBM has been resolved in the revised version of the MS. Prompted by the referees’ comments, we have expanded our work on Pol32 RBM phosphorylation (explained in more detail in response to Referee 1 (point 6) above). Briefly, we have performed in vitro kinase assays and found that, despite the presence of a predicted GSK3 family kinase consensus sequence within the Pol32 RBM, Pol32 is a poor phosphorylation substrate (new Supplementary Fig. S4; Rim11 likely impacts BIR through phosphorylation of the Ume6-Rpd3 histone deacetylase, please see Fig. A above). Focussing instead on the kinases that have been linked to bis-phosphorylation of Pol32 residues Thr256 and Thr257 (refs. 40-44 in the revised MS), we now demonstrate that Dbf4-dependent kinase (DDK) Cdc7 and Polo-like kinase (PLK) Cdc5 are capable of directly phosphorylating the Pol32 RBM (new Fig. 3A, B). The new data is described in the Results section entitled “The Pol32 RBM can be phosphorylated by multiple protein kinases.” (please see page 7, line 190-page 8, line 219). Importantly, the Pol32 T256A T257A bis-phosphorylation site mutant is not associated with a BIR efficiency defect, consistent with the phenotype of Pol32DRBM. However, upon deletion of the Pol32 PIP-box (Pol32_1-333 T256A T257A), cells show a significant drop in BIR efficiency as compared to cells expressing Pol32_1-333, indicating that phosphorylation at Thr256/257 in vivo allows cells to modulate BIR efficiency. Of note, Cdc7, which has previously been implicated in BIR efficiency by the Haber lab (Lydeard JR et al., Genes Dev. 24:1133-44. (2010) doi: 10.1101/gad.1922610), showed the strongest activity towards the Pol32 RBM in vitro. This might indicate that Pol32 high-affinity RBM binding is increasingly activated as cells transit S phase, potentially promoting effective BIR at broken replication forks or exogenous DNA breaks in S/G2 phase (please see Discussion, page 16, line 417-page 17, line 436 in the revised MS).\nConcerns\nThe analysis of phenotypes is relatively weak, as it relies on a single BIR assay that is highly inefficient, even in wild-type cells (only ~20% complete BIR). The differences between wild-type and mutants are within a ±5% range for some mutants. Additionally, several interesting predictions can be drawn from the proposed model that should be tested in vivo. A few additional experiments would significantly strengthen this work:\n1. Test BIR using another assay. The allelic BIR assay, which is commonly used, would be ideal. Decreased D-loop extension often leads to specific phenotypes, such as half-crossovers, which are expected in rim11Δ mutants.\nAuthors’ response: While the JRL-based system (developed by Jim Haber and co-workers: Lydeard JR et al., Nature 448:820-3 (2007) doi: 10.1038/nature06047), which we use throughout this MS, is robust and consistently identifies significant diqerences in BIR eqiciency in the tested mutants, we agree that independent validation of the key findings presented in Fig. 1 with an alternative assay can strengthen our data further. As suggested, we have now obtained the AM1003-based system from Prof Grzegorz Ira and have constructed additional reporter strains to measure allelic BIR as described by the Malkova lab (Deem et al., Genetics 179:1845-60. (2008) doi: 10.1534/genetics.108.087940). AM1003 is a haploid background with a second copy of chromosome III (MATa and truncated distal to the HOcs locus), which increases the efficiency of BIR repair off the uncleavable MATa-inc allele on the full-length copy of chromosome III (please see new Supplementary Fig. S1E; technical details added to the Methods section titled “BIR efficiency measurements”, page 18 of the revised MS). The two copies of chromosome III can be genetically distinguished because HML on the truncated copy is replaced by ADE1 while HML and HMR on the full-length chromosome III are replaced by ADE3 and HPH (which provides resistance to hygromycin), respectively. Deem and co-workers (and other authors since) have shown that approx. 80% of cells repair the DSB ensuing from galactose-induced HO endonuclease expression by BIR (generating ADE+ leu- cells) in this disomic system, while less than 10% repair by gene conversion (GC; ADE+ LEU+ survivors). In the presence of mutations which negatively affect BIR efficiency, chromosome loss and half-crossovers occur; this results in retention of the full-length copy of chromosome III only (the colonies are ade- leu- and red because loss of ADE1 on the truncated chromosome III disrupts the adenine biosynthesis such that red pigment accumulates) or fusions between the ADE1-containing fragment from the HO-endonuclease cleaved chromosome III and the distal part of the full-length copy (ADE3 is lost; white ade- leu- colonies), respectively. In good agreement with Deem and co-workers, we observe that chromosome loss events and half-crossovers are exceedingly rare in the wild-type, but their frequency is significantly increased in pol32D-mutant cells. Importantly, and confirming our truncational analysis of POL32 in the ectopic JRL-based BIR assay system, we find that chromosome loss and half-crossovers occur much more frequency compared to wild-type upon removal of PIP box (pol32_1-333) and that further truncation (pol32_1-249) – thus removing the PIP and RBM modules – results in a phenotype very similar to that observed for pol32D cells (new Supplementary Fig. S1F). These results, which corroborate our conclusion that both the PIP-box and RBM of Pol32 promote D-loop DNA synthesis and thereby BIR efficiency, are described in the revised version of the MS on page 4, line 106-page 5, line 115:\n“Next, we analysed DSB repair in an independent tester strain which is disomic for chromosome III (with one copy harbouring an HO-cut site and the other serving as repair template)36. In this system, DSB repair is highly efficient, and cells overwhelmingly use BIR with a small contribution of DSB repair by gene conversion. In contrast, pol32D cells show reduced BIR efficiency, a strong increase in chromosome loss events, and the formation of half-crossovers indicative of failed DSB repair and aberrant processing of BIR intermediates, respectively.36 We observed similar levels of Pol32-independent BIR, chromosome loss, and half-crossovers in cells harbouring a POL32 deletion or a truncation of Pol32 at residue 249 (Supplementary Fig. S1E and F). Thus, Pol32_1-249 results in a Pol32-null phenotype for ectopic and allelic interhomolog BIR.”\n2. Measure BIR kinetics in an efficient BIR assay. It would be informative to examine BIR kinetics in mutants such as pol32_RBM, rim11Δ, and pol32 phosphorylation mutants. In particular, pol32_RBM might exhibit a BIR delay.\nAuthors’ response: We agree with Referee 2 that measurements of BIR kinetics provide an excellent possibility to uncover BIR defects not easily detected in an end-point DNA double-strand break repair assay. To assess BIR kinetics, we used the PCR-based assay established by the Haber lab (Lydeard JR et al., Nature 448:820-3 (2007). doi: 10.1038/nature06047; described in a new Methods section on page 19 titled “BIR kinetics measurements” in the revised version of the MS). Using primers for the amplification of the nascent CAN1 gene that is newly formed through BIR after HO-cut induction, we detected a marked delay in repair kinetics in the pol32_1-133 RBM* mutant, where inactivation of the RBM is combined with a deletion of the PIP box, compared to a pol32_1-133 mutant. This is consistent with the loss in BIR eqiciency seen for the pol32_1-133 RBM* mutant. Interestingly, and as anticipated by this referee, we could even detect evidence of perturbed repair kinetics in pol32_RBM* cells, which corroborates that the Pol32 RBM contributes to promoting more stable D-loop DNA synthesis, even in the context of full-length Pol32. The data obtained with the RBM* mutants is presented in new Fig. 2E and described in the text on page 7, lines 181-188:\n“A PCR-based BIR assay23 for the detection of new DNA synthesis initiated at the centromere-proximal side of the HO cut site (see Supplementary Fig. S1B) upon invasion of the repair template showed that Pol32 RBM* was associated with a slight but reproducible delay in the formation of BIR repair intermediates in the first 8 h after DSB induction. While the levels of BIR product formation mediated by Pol32 RBM* were eventually indistinguishable from wild-type, Pol32_1-333 RBM* was associated with a prolonged BIR DNA synthesis defect that grew more pronounced between 8 and 20 h after DSB induction (Fig 2E). Together, these data indicate that Pol32 RBM-RPA interactions promote BIR.”\n3. Examine crossover frequency in the Mph1 C-terminal mutant. The mph1ΔC (1-954) mutant shows reduced interaction with RPA and stimulates BIR, likely due to loss of its effect on D-loop unwinding. A key prediction is that this mutant should exhibit a higher level of crossover outcomes, which should be tested.\nAuthors’ response: To test whether Mph1 relies on its newly defined RBM for the destabilization of D-loops, we made use of the afore-mentioned (please see response to point 2 above) PCR-based kinetic assay. We found that deletion of the Mph1 RBM resulted in faster BIR repair kinetics, i.e., an accelerated de novo formation of CAN1 repair intermediates through D-loop DNA synthesis after HO-cut induction. This is consistent with the expectation that Mph1-RPA interactions support Mph1-dependent D-loop unwinding, thus facilitating Mph1’s role as a BIR antagonist. This data is presented in new Supplementary Fig. S8D in the revised version of the MS, and is referred to in the text on page 12, lines 323-327:\n“Cells expressing the truncated Mph1_1-954 from the endogenous locus exhibited significantly elevated BIR eIiciency, phenocopying a complete deletion of MPH1 (Fig. 4E). Consistently, BIR intermediate formation was higher in mph1_1-954 cells compared to MPH1 wild-type control cells as measured by PCR23 6 h after DSB induction (Supplementary Fig S8D).”\n4. Test the genetic interaction between Rim11 and Mph1. If Rim11-mediated phosphorylation of Pol32 enhances Polδ-RPA interaction and reduces the negative impact of Mph1-RPA on BIR, then rim11Δ mph1-954 double mutants should not exhibit any defects in BIR. Testing BIR efficiency in this double mutant would directly test the authors' model.\nAuthors’ response: This specific experiment is superseded by our new findings that DDK and PLK, which have been implicated in vivo (and not Rim11) mediate the direct phosphorylation of the Pol32 RBM in vitro. The essential and pleiotropic nature of Cdc7 and Cdc5 preclude the suggested experiment. However, we have extended our analyses of the Pif1 and Mph1 RBMs. Sequence alignment and AlphaFold modelling shows that the Pif1 and Mph1 RBMs contain pre-existing negative charges (Asp71 and Asp990, respectively) equivalent to Pol32 Thr256/257 bis-phosphorylation. This allows Pif1 and Mph1 to interact with the Rfa1 NTD similarly to the Pol32 pRBM by engaging a small hydrophobic pocket on the surface of Rfa1 independently of phosphorylation (please see new Supplementary Fig. S7E, F). As an example in point, we demonstrate that Pif1 RBM phosphorylation has minor effects on RPA affinity and no significant impact on BIR efficiency (new Supplementary Fig. S8 A, B). Because Mph1 requires its own RBM and competitive access to the primer junction engaged by Pol d during BIR to mediate D-loop disassembly, these results strongly suggest that Pol d with a phosphorylated Pol32 subunit may a greater obstacle to Mph1 than the non-phosphorylated form. We refer to these consideration on page 15 (from line 405) in the revised version of the MS:\n“Mph1 targets extended D-loops24,49 and dissociates the nascent strand47 to restrict BIR efficiency48,60. We find that these actions of Mph1 depend on a newly defined RBM at the Mph1 C-terminus. The Mph1 RBM binds RPA more avidly than the non-phosphorylated Pol32 RBM, but with similar affinity to the phosphorylated form. It is therefore tempting to speculate that Pol32 RBM phosphorylation may help Pol d to locally resist the actions of Mph1 at the D-loop primer junction. Thus, Pol32 RBM phosphorylation may promote BIR by stabilizing Pol d at the primer junction and mitigating D-loop collapse. As a consequence, the risk of chromosome aberrations5,6 arising from ectopic recombination upon premature release of the nascent strand and invasion at a non-contiguous site with sequence homology61-64 could be reduced. Conversely, Pol32 RBM de-phosphorylation may favour D-loop disassembly (…).”\nMinor Comments:\nRim11 localization and function: Rim11 is typically localized in the cytoplasm. Is it known to phosphorylate any other nuclear proteins? Is there evidence that Rim11 directly phosphorylates Pol32?\nAuthors’ response: While there is precedent for the Rim11-dependent phosphorylation of nuclear factors (Rubin-Bejerano I et al., Mol Cell Biol 24:6967-79 (2004). doi: 10.1128/MCB.24.16.6967-6979.2004; Malathi K et al., Mol Cell Biol 17:7230-6 (1997). doi: 10.1128/MCB.17.12.7230), we have now tested the ability of Rim11 to directly phosphorylate Pol32. As detailed above, our new experiments show that under conditions where Rim11 is active, the kinase fails to phosphorylate the Pol32 RBM in vitro (new Supplementary Fig. S4). Instead, we identify the Pol32 RBM as a direct target of DDK (Cdc7) and PLK (Cdc5) (new Fig. 3A, B). Cdc7 has previously been implicated in BIR efficiency (Lydeard JR et al., Genes Dev. 24:1133-44. (2010) doi: 10.1101/gad.1922610).\nClarification in Lines 39-41: The work first demonstrated that BIR is limited in the repair of replication fork breakage—PMID: 26273056.\nAuthors’ response: In the revised version of the MS, we have amended the text to highlight the specific findings reported in PMID: 26273056 (this is ref. 2 in the original and the revised version of the MS) to read (on page 2, lines 41-44):\n“(…) This can be achieved through active D-loop processing by structure-specific nucleases and the arrival of a converging DNA replication fork.2,9 Alternatively, cells may suppress BIR to favour the generation of a two-ended DSB and subsequent repair by error-free homologous recombination.10-12”\nReviewer #3 (Remarks to the Author):\nIn the manuscript by Jones and co-workers, the authors report the identification of a novel RPA binding motif (RBM) in the Pol32 subunit of the DNA polymerase Delta using the budding yeast as a model organism. Normally, the Pol32 recruitment to DNA is controlled through its interaction with PCNA via a PCNA-interacting peptide (PIP) located at the C-terminus of Pol32. The importance of the identified RBM in vivo, based on DNA repair by BIR, could only be revealed if the Pol32-PCNA interaction is eliminated from the system by the Pol32 C-terminal deletion involving the PIP, as shown by the experiments in figure 2D. The RPA-Pol31-Pol32 in vivo interaction is detected by protein CoIPs (Figure 2C).\nThe in vitro binding experiments and Alpha-fold modelling support the idea that phosphorylation of some S/T residues within the RBM motif strengthens the RPA-Pol32 interaction. The phosphor-site mutations have an expected effect on BIR in the background where the Pol32-PCNA interaction is lost, but behave in a confusing way in full length Pol32. The authors then claim that Rim11 is the kinase responsible for the phosphorylation of the RBM in Pol32.\nFinally, a structural parallel is drawn to the RBMs from Pif1 and Mph1, two other proteins known to be involved in BIR and carrying RBMs.\nI have two concerns about this manuscript which I would like to discuss separately:\n1. the suitability for publishing it in Nature Communications;\n2. the experimental part.\nAs presented, the focus of the manuscript is on identifying a novel RBM in Pol32. Based on the fact that this is just another RBM added to the several previously reported in different organisms, including some of those in budding yeast, the novelty of this manuscript doesn’t advance the field enough to grant a publication in NC, particularly since the biological significance of this RBM in Pol32 is unclear and its potential regulation through phosphorylation is not studied well enough. Based on the experiments in this manuscript, the RBM plays a secondary role to the PIP in the Pol32 recruitment in BIR: with the Pol32 PIP being functional, the RBM has no effect on the efficiency of BIR. The RBM may play a more important role in some other genome stability aspect independent of BIR, but it is not presented in this work and, therefore, it remains unclear how important it is.\nWith this said, I could see how the focus of the manuscript could be shifted to a more of a big-picture question, to stress the novelty of identifying extended RBMs where phosphorylation of the residues adjacent to the hydrophobic cores plays a role in genome stability via stimulating the recruitment of the repair proteins to DNA. This would be a major revision and require a lot of additional experiments, such as analysing multiple RBMs, the ones in Pif1 and Mph1 at the very least, demonstrating the phosphorylation dependence on the DNA damage response (or other trigger) in vivo, different forms of peptide binding to RPA similar to those for Pol32 in figure 4A, plus addressing the physiological role of RBM phosphorylation in BIR through analysing the phosphosite mutants in the genetic BIR assays. These experiments would then justify the model proposed by the authors in Figure 5 and might make the story conceptually novel enough for NC to consider. As is, there is very little experimental data to support this model: the phosphorylation of RBM in Pol32 is not important for BIR in vivo; the phosphorylation of RBMs in Pif1 and Mph1 hasn’t been addressed experimentally. Also, it is not known what triggers the RBM phosphorylation – cell cycle, DNA damage signalling, etc. Therefore, the model in figure 5 is based on computer modelling and imagination, but not supported by sufficient experimental data.\nAuthors’ response: While we discover a novel RBM that contributes to the function of Pol32 in BIR, we agree with referee 3 that important significance and novelty arises from the first description of a mechanism by which RBMs can be activated by phosphorylation for high-affinity PRA binding. In our major experimental revision, we have included a number of experiments to solidify our model presented in revised Fig. 5. Specifically, we provide additional evidence for the biological significance of the Pol32 RBM through BIR kinetic measurements (new Fig. 2E), identify the Pol32 RBM as a direct phosphorylation target of Dbf4-dependent kinase (DDK) Cdc7 and Polo-like kinase (PLK) Cdc5 using in vitro kinase assays and analysis by mass spectrometry (new Fig. 3A, B and Supplementary Fig. S4), validate the importance of Pol32 RBM phospho-residue-Rfa1 NTD interactions by mutational analysis of Rfa1 (new Supplementary Fig. S6A, B), and investigate the RBMs of Pif1 and Mph1 in more detail (new Fig. 4 A-C, E, new Supplementary Fig. S7E, F and new Supplementary Fig. S8A, B, D).\nWe used a PCR-based assay (Lydeard JR et al., Nature 448:820-3 (2007). doi: 10.1038/nature06047) to measure BIR kinetics by following the formation of BIR intermediates at the cleaved HOcs locus. As expected, there was a marked delay in BIR repair in pol32_1-133 RBM* cells. Interestingly, we could also detect evidence of delayed repair kinetics in pol32_RBM* cells, which supports the role of Pol32 RBM in promoting D-loop DNA synthesis, even in the context of full-length Pol32. This data is presented in new Fig. 2E and described in the revised version of the MS on page 7, lines 181-188:\n“A PCR-based BIR assay23 for the detection of new DNA synthesis initiated at the centromere-proximal side of the HO cut site (see Supplementary Fig. S1B) upon invasion of the repair template showed that Pol32 RBM* was associated with a slight but reproducible delay in the formation of BIR repair intermediates in the first 8 h after DSB induction. While the levels of BIR product formation mediated by Pol32 RBM* were eventually indistinguishable from wild-type, Pol32_1-333 RBM* was associated with a prolonged BIR DNA synthesis defect that grew more pronounced between 8 and 20 h after DSB induction (Fig 2E). Together, these data indicate that Pol32 RBM-RPA interactions promote BIR.”\nAs explained in more detail in our response to specific point 4 below, we have turned to in vitro phosphorylation assays to investigate the kinase(s) responsible for Pol32 RBM phosphorylation. Despite the presence of a predicted consensus phosphorylation site, Pol32 RBM turned out to be a poor substrate for Rim11 (new Supplementary Fig. S4; the impact of Rim11on BIR may be explained by its role in phosphorylating the Ume6-Rpd3 histone deacetylase, which in turn modifies and alters the activity of Pif1 (Ononye OE et al., J Biol Chem 295:15482-15497 (2020). doi: 10.1074/jbc.RA120.015164); please see our response to Referee1, point 6; and Fig. A above). We next focussed on the kinases linked to phosphorylation of Pol32 in vivo (phospho-proteomic studies in refs. 40-44 in the revised version of the MS). We were able to demonstrate that Cdc7 and Cdc5 are capable of directly phosphorylating the Pol32 RBM (new Fig. 3A, B). This suggests a cell-cycle stimulus where phosphorylation-dependent high-affinity Pol32 RBM binding to the Rfa1 NTD is increasingly activated as cells progress through S phase, which would promote BIR at broken replication forks in S/G2 phase (please see Discussion, page 16, line 417-page 17, line 436 in the revised MS).\nFollowing Referee 3’s suggestion, we have further investigated the Pol32, Pif1 and Mph1 RBMs (note that we have validated that the C-terminus of Mph1 contains a bona fide RBM in the revised version of the MS, please see our response to specific point 5 below). Testing a mutant Rfa1 NTD protein with Arg91Ala and Lys95Ala substitutions and the pThr257 or pThr256 pThr257 (bis-)phosphorylated Pol32 RBM, we observed that the effect of phosphorylation in promoting high-affinity binding was neutralized (new Supplementary Fig. 6A, B). This provides important experimental validation of the significance of the modelled pRBM-Rfa1 contacts with Rfa1 Arg91 and -Lys95 RBM in the pRBM kinked conformation that allows Leu262 to project into a well-characterized hydrophobic ‘side-pocket’, thus contributing to interaction strength with RPA (please see revised Fig. 3D). We further demonstrate that both, the Pif1 and Mph1 RBMs, do not require phosphorylation for high-affinity Rfa1 NTD binding. In contrast to the Pol32 RBM, phosphorylation of the Pif1 RBM peptide (pSer70 and pSer72) had a mild impact on Rfa1 NTD binding affinity (improving the already low K determined by FP from 0.32 µM for the non-phosphorylated form to 0.15 µM; new Supplementary Fig. S8A). Concomitantly, cells expressing Pif1 S70D S72D did not show increased BIR efficiency (new Supplementary Fig. S8A). Sequence alignment and AlphaFold modelling shows that the Pif1 and Mph1 RBMs contain pre-existing negative charges (Asp71 and Asp990, respectively) at positions equivalent to Pol32 Thr256 and Thr257. This implies that the Pif1 and Mph1 RBMs might interact with the Rfa1 NTD in a high-affinity configuration similar the phosphorylated Pol32 RBM, explaining their phosphorylation-independent avid binding of the Rfa1 NTD. In support of this view, we provide modelling showing that the phosphorylation is not required for the Pif1 and Mph1 RBMs to engage the small hydrophobic ‘side-pocket’ on the surface of Rfa1 via Trp75 and Phe993, respectively (new Supplementary Fig. S7E, F). Since overall BIR efficiency is determined by the interplay of agonists (such as Pol32) and antagonists (Mph1), we suggest that the dynamic regulation of one effector (Pol32) is sufficient to fine-tune BIR outcomes (page 15, from line 405 of the revised version of the MS):\n“Mph1 targets extended D-loops24,49 and dissociates the nascent strand47 to restrict BIR efficiency48,60. We find that these actions of Mph1 depend on a newly defined RBM at the Mph1 C-terminus. The Mph1 RBM binds RPA more avidly than the non-phosphorylated Pol32 RBM, but with similar affinity to the phosphorylated form. It is therefore tempting to speculate that Pol32 RBM phosphorylation may help Pol d to locally resist the actions of Mph1 at the D-loop primer junction. Thus, Pol32 RBM phosphorylation may promote BIR by stabilizing Pol d at the primer junction and mitigating D-loop collapse. (…) Conversely, Pol32 RBM de-phosphorylation may favour D-loop disassembly (…).”\nOur extended work on RBMs in the key components of the BIR machinery thus shows they exist in two flavours, either supporting phospho-regulated or constitutive high-affinity Rfa1 NTD binding. This raises the question whether the mechanism of phospho-activated pRBM-Rfa1 NTD that we describe for Pol32 captures a general phenomenon. Based on sequence analysis, we predict it does, since RBMs with potential phosphorylation sites (serines and threonines) or phospho-mimetic residues at positions equivalent to Thr256 and Thr257 in Pol32 exist. Excitingly, whilst our manuscript was under review, a complementary study was published in Nature Communications: Noto A et al., Nat Commun 16:997 (2025) doi:10.1038/s41467-025-55958-z) that showed CK2-mediated phosphorylation of an acidic domain in WRN promoted RPA association in human cells, and this facilitated the recovery of stalled DNA replication forks. The authors observed that phosphorylation of a tandem RBM within the WRN acidic domain (at Ser440 in the first RBM and Ser467 in the second) enabled RPA association in cells, while this was not observed with a non-phosphorylatable mutant. There is no structural basis for phospho-regulated RBMs in WRN, but this can now be rationalized when considering our study on the phospho-regulated RBM in Pol32. Indeed, when we predict the corresponding phosphorylated RBM in WRN (pSer440) with RPA70-NTD, the structural basis for the interaction is analogous to Pol32 (Fig. B, panels a and b herein). Similarly to the Pol32 RBM (Fig. B, panel c), the unmodified WRN-RBM adopts a linear a-helix, which is the configuration found in the X-ray crystal structure of this RBM associated with RPA70-NTD (PDB 7XUT) (Fig. B, panel d). Upon phosphorylation, the continuous a-helix of the Pol32-RBM is ‘broken’ and engages in new interactions with the Rfa1 NTD groove (as described above, pThr257 or pThr256 in the bis-phosphorylated RBM engage with the side chains of Rfa1 Arg91 and -Lys95 and the kinked conformation allows Leu262-hydrophobic ‘side-pocket’ interactions; please see revised Fig. 3D). AlphaFold structural predictions of a phosphorylated WRN-RBM with RPA70-NTD suggest a highly related conformational change (Fig. B, panels c and d). This is all the more remarkable because Rfa1 and RPA70 only share 20% sequence identity.\nTherefore, our molecular dissection of Pol32 RBM phosphorylation, which enhances RPA-association 150-fold, represents an exemplar case for a phospho-regulated RBM, providing a molecular mechanism which we anticipate will be much more widely applicable. Current evidence supports the view that RBM phosphorylation is deployed by cells to enhances RPA interaction and regulate DNA repair outcomes in vivo in at least two pathways: BIR (our study) and fork recovery (please see Noto A et al., Nat Commun 16:997 (2025) doi:10.1038/s41467-025-55958-z.) Thus, while the focus of our work is the mechanism of DSB repair by BIR, shedding important new light on the enigmatic ability of Pol32 to modulate BIR repair, it provides a paradigm for a hitherto undescribed control mechanism for DNA metabolic processes involving RPA-ssDNA intermediates. This provides the basis for exciting future work in experimentally cataloguing the effects of RBM phosphorylation across RPA client proteins and identifying the biological processes regulated by phospho-dependent Rfa1/RPA70 NTD binding from yeast to human. This broad relevance is now referred to in the Discussion in the revised version of the MS (page 17, lines 441-449):\n“More generally, the phospho-activated RBM within Pol32 provides a paradigm for dynamic control of protein association with the Rfa1 NTD basic-hydrophobic groove and hydrophobic pocket to fine-tune DNA repair. Consistent with this notion, phospho-dependent interactions of the Werner syndrome helicase (WRN) with RPA have recently been reported to promote DNA replication fork recovery.66 With RPA being involved in virtually all DNA transactions during replication, repair, and recombination involving ssDNA, and a growing number of identified Rfa1/RPA70 client proteins14, pRBM-Rfa1/RPA70 interactions could be broadly relevant in genome maintenance.”\nThe experimental part:\n(1) Figure 1F. The authors use HU to assay different POL32 alleles. The rest of the paper is focused on BIR, i.e. DSB repair involving activation of the G2/M checkpoint rather than an intra-S phase checkpoint triggered by HU. It might be informative to add a spot test using Phleomycin or a similar drug inducing DSBs rather than stalled forks.\nAuthors’ response: The Pol32 N-terminal domain is required for Pol d complex stability and loss of POL32 results in cellular sensitivity to a wide range of replication-stress inducing agents including HU, MMS, and 4NQO (e.g., Karras GI & Jentsch S, Cell 141:255-67 (2010) doi: 10.1016/j.cell.2010.02.028). This can be overcome by artificially stabilizing the Pol d complex in pol32D cells; the BIR defect of pol32D cells, however, cannot be overcome in this way (Shimada K et al., Life Sci Alliance 4:e202101138 (2021) doi: 10.26508/lsa.202101138). This indicates that BIR requires features of Pol32 outside the N-terminal domain, which we show include the newly identified C-terminal RBM. In Fig. 1F, we demonstrate that truncation of the C-terminal features of Pol32 which support BIR (PIP-box, DPIM and RBM; please see Fig. 1E) do not sensitize cells to replication stress sensitivity, indicating that Pol d complex formation is intact and Pol d remains DNA replication competent. We have now performed a spot assay on phleomycin-containing medium and the results are similar to those obtained with HU; pol32D cells show hypersensitivity while Pol32 C-terminal truncation mutations are associated with very little to no apparent sensitivity. This is consistent with DNA damaging drugs such as phleomycin causing replication stress and results showing that POL32 is not required for DSB repair by gene conversion (Lydeard JR et al., Nature 448:820-3 (2007). doi: 10.1038/nature06047), the preferred pathway of repair at exogenously induced DSBs. The phleomycin data (Fig. C herein) is available, but we suggest inclusion in the revised MS would not add significantly to the HU sensitivity assay demonstrating that Pol32 C-terminal deletions affecting BIR do not result in pol32D-linked replication stress sensitivity.\n(2) Figure 2C. My understanding is that the C-terminal truncation form of Pol32 used in this experiment doesn’t have either RBM or PIP. It would be much more informative to use an RBM-only deletion mutant instead and present a similar experiment later in the paper when the RBM is identified. A separate point – if the authors suggest that the RBM might be phosphorylated to facilitate the repair of DNA damage (Figure 5), then it would make sense to do a pull-down from the cells with and without DNA damage induced.\nAuthors’ response: We fully concur with Referee 3 that deploying an RBM-only internal deletion to assess the role of Pol32 in the newly found Pol d complex-RPA interaction is more elegant than a Pol32 C-terminal tail deletion mutant. In our revisions, we have constructed the relevant strains and repeated the experiment shown in former Fig. 2C. Using cells harbouring TAP-tagged Pol31 and FLAG-tagged Pol32 DRBM, we demonstrate that an internal deletion of the Pol32 RBM strongly diminishes the recovery of RPA observed with wild-type Pol32 to nearly background levels (new Fig. 2B in the revised version of the MS). This confirms our previous conclusion that Pol d-RPA interactions depend on the newly identified RBM within Pol32 (referred to on page 6, line 162-page 7, line 166):\n“However, an internal deletion of Pol32 aa 247 to 258 (hereafter referred to as DRBM) diminished Pol d-RPA interactions (Fig. 2B). These data are therefore consistent with the newly identified acidic-hydrophobic motif within Pol32 functioning as a bona fide RBM and serve to confirm the novel Pol d-RPA interaction.”\nBIR-dependent D-loop DNA synthesis does not require checkpoint activity (Malkova A et al., Mol Cell Biol 25:933-44 (2005) doi: 10.1128/MCB.25.3.933-944.2005; Kimble MT et al., Mol Cell 85:61-77.e6 (2025) doi: 10.1016/j.molcel.2024.10.032.), and there is currently no in vivo evidence to suggest that Pol32 phosphorylation is controlled by the DNA damage checkpoint. Instead, we followed the evidence provided by phospho-proteomic studies and now provide new data showing that DDK and PLK can directly phosphorylate the Pol32 RBM (please see our response to specific point 4 below), suggesting that the alternative stimulus suggested by Referee 3, cell-cycle progression, is a driver of Pol32 RBM phosphorylation.\n(3) Figure 3A. The BIR for T257A is very confusing. The allele acts as dominant negative and the authors suggest a somewhat speculative explanation in the discussion. My suggestion would be to look for another RBM in Pol-delta as the mutated one in Pol32 might be blocking the access to an alternative RBM elsewhere in Pol-delta. In Figure 2C, the pulldown of Pol31 brings some RPA even in the absence of the C-terminus of Pol32 (last lane in the blot) suggesting another interaction point between Pol-delta and RPA.\nAuthors’ response: We thank Referee 3 for their thoughtful remarks here, and in the subsequent specific point 4, regarding the T257A BIR phenotype. We also have considered the possibility of an intramolecular dominant negative effect associated with this mutation. Within Pol32, we cannot identify another RBM (Supplementary Fig. S2) and our extensive new AlphaFold 3 modelling with the entire Pol d-PCNA-DNA-Rfa1-NTD assembly (please see Referee 1, point 1) exclusively identifies the Pol32 RBM-Rfa1 interaction for Pol d, which is consistent with a significant drop in RPA recovery by Pol d pull-down to background levels when the Pol32 RBM is internally deleted (new Fig. 2B in the revised version of the MS). However, the comments have prompted us to fully revisit the question of Pol32 phosphorylation and the behaviour of phospho-null mutants. It is true that a negative effect of phospho-null mutations within the Pol32 RBM are unexpected because the Pol32 DRBM mutant demonstrates that RBM phosphorylation is not required for BIR efficiency as measured in the genetic end-point assay (Fig. 2D). Furthermore, our assessment of kinases implicated by phospho-proteomic studies in Pol32 RBM phosphorylation in vivo during this revision (please see our reply to specific point 4 below) shows that multiple kinases can directly phosphorylate the RBM in vitro at Thr256, Thr257, and Ser261. Concentrating solely on Thr257 phosphorylation (which was the first identified and only annotated RBM phospho-site on SGD at the start of this project and conforms to a potential GSK3 kinase target site) has therefore proven too narrow an approach. Strikingly, the puzzling dominant negative effect of the T257A mutation was no longer observed when we combined T257A with T256A (please see new Fig. 3F in the revised version of the MS). This indicates a spurious effect associated with Pol32 T257A, potentially as a result of alterations to the normal Pol32 phospho-regulation. In the revised version of the MS, we have therefore eliminated the T257A mutant. Our new experiments are now aligned with the in vivo evidence showing phosphorylation of Pol32 being predominantly at Thr256 and Thr257 (see also specific point 4 below) and resolve the previously puzzling discrepancy between the Pol32 DRBM and Pol32 T257A mutants. The data is referred to in the revised version of the MS on page 10, line 270-page 11, line 283:\n“Addressing the impact of enhanced Rfa1 NTD interactions mediated by Pol32 pRBM on BIR, we confirmed that phospho-mimetic amino changes Thr256Asp and Thr257Asp within the RBM increase the affinity for Rfa1 (Supplementary Fig. 6C). We then generated versions of the ectopic BIR reporter strain expressing Pol32 T256D T257D, which was present at concentrations comparable to wild-type Pol32 (Fig. 3E). Importantly, Pol32 T256D T257D boosted BIR efficiency as compared to the control strain, identifying a first Pol32 mutant that improves BIR (Fig. 3F). Like Pol32 DRBM and -RBM*, an endogenously expressed Pol32 T256A T257A phospho-null mutant was not associated with a BIR efficiency defect (Fig. 3F). However, as in the case of Pol32_1-333 RBM*, a strain expressing Pol32_1-333 T256A T257A showed a significant drop in BIR efficiency, suggesting that phosphorylation at Thr256/257 is required for BIR efficiency in the absence of the Pol32 PIP-box. Consistently, T256D/T257D continued to enhance BIR efficiency in the context of a Pol32 PIP-box truncation mutant (Fig. 3G). These data are consistent with the notion that the phosphorylation of the Pol32 RBM in vivo40-44 can be deployed by cells to modulate BIR efficiency.”\n(4) Figure 3C-D. It is very puzzling that the authors didn’t consider Chk2 kinases (Rad53 and Dun1) as the candidates for the Pol32 phosphorylation, particularly in the light of their model in Figure 5 and the fact that BIR strongly depends on Rad9 involved in Rad53 activation.\nAuthors’ response: The Haber lab has previously demonstrated that Rad9 promotes BIR-dependent survivors after an induced HO-cut. However, the execution of BIR repair by D-loop DNA synthesis mediated by Pol d is not dependent on Rad9 or checkpoint activation. Thus, BIR is restored in rad9D cells upon nocodazole arrest and occurs with normal kinetics. It is therefore the Rad9-dependent establishment of cell-cycle arrest – providing time for BIR to occur – not the action of DNA damage response kinases on the BIR machinery that explains the requirement for Rad9 for BIR efficiency (Malkova A et al., Mol Cell Biol 25:933-44 (2005) doi: 10.1128/MCB.25.3.933-944.2005). The mechanistic independence of BIR from checkpoint kinases is consistent with recent data from Lorraine Symington’s lab showing that replication-coupled DSBs are repaired by BIR without activation of the checkpoint (Kimble MT et al., Mol Cell 85:61-77.e6 (2025) doi: 10.1016/j.molcel.2024.10.032.). In our renewed efforts to identify bona fide Pol32-phosphorylating kinases we have therefore not concentrated on checkpoint kinases but all kinases for which data exists in the literature implicating them in the phosphorylation of Pol32 RBM residues in vivo, include Cdc7 (DDK), Cdc5 (PLK), and Cyclin-dependent kinase (CDK) Cdc28 (please see below for details).\n(4 cont’d) The genetics presented in [Fig. 3] panels C and D is not easy to interpret conclusively, mainly because the T257A mutation in the full length Pol32 context has a hard-to-explain phenotype (Figure 3A, see above). However, T257A behaves much more predictably in Pol32(1-333). This is why the genetics should be done in this background by comparing BIR in Pol32(1-333), Pol32(1-333) Rim11-deletion, Pol32(1-333)-T257A Rim11-deletion, Pol32(1-333) T257D Rim11-deletion. If the genetic experiments still hold Rim11 relevant (it will only work if Rim11 has Pol32 as the only target in BIR), a physical interaction should be addressed between Pol32 (or Pol-delta) and Rim11. As presented, the evidence for the regulation of Pol32 by Rim11 is not convincing and unless more data are added, this part should be excluded from the manuscript.\nAuthors’ response: Following on from the reply to specific point 3 above detailing that the previously puzzling reduction in BIR efficiency observed for the Pol32 T257A mutant is not seen for a Pol32 T257A T256A double mutant and thus not a consequence of loss of phosphorylation at these sites but a spurious dominant negative effect, we have extended our analysis of potential kinases mediating Pol32 RBM phosphorylation. We can now rule out Rim11 as the kinase responsible for Pol32 Thr257 phosphorylation and have taken the lead from in vivo data (refs. 40-44 in the revised version of the MS) demonstrating phosphorylation of the Pol32 RBM to identify the Pol32 RBM as a direct phosphorylation target of Cdc7 and Cdc5.\nPrompted by Referee 1, we have taken an in vitro kinase assay approach. This showed that Rim11 is not capable of phosphorylating the Pol32 RBM despite the presence of a predicted GSK3 family kinase consensus sequence (new Supplementary Fig. S4). However, deletion of UME6, a known substrate of Rim11 (Malathi K et al., Mol Cell Biol 17:7230-6 (1997). doi: 10.1128/MCB.17.12.7230) and regulator of Pif1 (Ononye OE et al., J Biol Chem 295:15482-15497 (2020). doi: 10.1074/jbc.RA120.015164) phenocopies the BIR defect of rim11D cells (please see Fig. A above).\nTo identify bona fide Pol32 RBM kinases, we now considered the kinases for which evidence exists that they promote Pol32 phosphorylation at residues within the RBM in vivo. The Pol32 RBM contains three potential phosphorylation sites residues Thr256, Thr257, and Ser 261. Phospho-proteomics have shown that the Pol32 RBM is predominantly modified at both threonine residues in vivo (ref. 40-44 in the revised version of the MS). These studies have implicated multiple kinases including Cdc7, Cdc5, and Cdc28. We also considered the acidophilic casein kinase II (CKII) given the acidic-hydrophobic nature of the RBM amino acid composition. TAP-tagged versions of these kinases were affinity-captured and tested for activity on Pol32 RBM fragments (aa 248 to 264) fused to thioredoxin. Cdc7, Cdc5, and CKII were able to phosphorylate the Pol32 RBM fragment while Cdc28 (or affinity captured cyclins Clb1, 3, or 5) and Rim11 showed no activity on Pol32 (new Supplementary Fig. 4A, B). Phosphorylation levels dropped when Cdc7, Cdc5, and CKII were tested on a version of the RBM fragment with phospho-null mutations T256A/T257A (new Supplementary Fig. 4B). To map phosphorylation events, we performed mass spectrometry, revealing that while all affinity-purified kinases exerted detectable kinase activity on the thioredoxin fusion, the Pol32 RBM was only phosphorylated by Cdc7, Cdc5, and CKII (new Fig. 3A, B and new Supplementary Fig. 4C). As expected, the acidophilic kinases showed the most robust activity on the Pol32 RBM, and Cdc7 has been reported to be critical for BIR efficiency (Lyderad JR et al., Genes Dev 24:1133-44 (2010). doi: 10.1101/gad.1922610). The existing in vivo data (refs.40-44), combined with our new in vitro data, suggest Cdc5, Cdc7, and possibly additional acidophilic kinases such as CKII, contribute to laying down the appropriate ‘phospho-code’ on Pol32. Our experiments are described in new Results section entitled “The Pol32 RBM can be phosphorylated by multiple protein kinases.” (starting on page 7 of the revised version of the MS). We consider the findings in the Discussion on page 16, line 417-page 17, line 436:\n“Regulation of the Pol32 RBM phosphorylation status may involve multiple kinases. Ddc2 RBM Ser 10 and Ser11 phosphorylation is driven by the constitutively active acidophilic kinase CKII and its regulation is likely to involve the actions of phosphatases.37 Consistent with the sequence similarity between the Ddc2 and Pol32 RBMs, we find that CKII can also phosphorylate the Pol32 RBM in vitro and may therefore contribute to the phospho-regulation of Pol32. Several other kinases have been implicated in Pol32 RBM phosphorylation in vivo. 40-44 We confirmed the ability of Cdc5 (PLK) and Cdc7 (DDK) to directly phosphorylate the Pol32 RBM in vitro. Phospho-proteomics suggest that Cdc7 phosphorylates Ser and Thr residues with Asp/Glu/pSer/pThr at the plus 1 position44, and bis-phosphorylation at Thr256/257 within Pol32 conforms with this preference. Interestingly, Cdc7 showed the most robust activity towards the Pol32 RBM in vitro and has previously been reported46 to promote BIR in vivo. DDK is active from the start of S-phase through to mitosis, while PLK is activated at the G2/M transition. Both kinases act to align cellular processes with cell cycle stage and are known to co-regulate recombination-associated reactions, for example by coordinating the resolution of recombination intermediates with mitotic entry65. Their activity towards Pol32 would suggest that the Pol32 RBM becomes increasingly activated for high-affinity RPA binding as cells progress through S phase, which could promote effective BIR at broken replication forks in S/G2 phase. However, the details of Pol32 phospho-status regulation, which may involve additional kinases as well as phosphatases, remain to be elucidated in future studies.”\nWe appreciate the insistence of Referee 3 on this point, which has resulted in much more clarity and alignment with existing data on the point of Pol32 RBM phosphorylation. We feel that the identification of bona fide direct modifier kinases of the Pol32 RBM is an important addition to the revised MS.\n(5) Figure 4C. The C-terminal deletion (1-953) truncates way too much to make a conclusion about the relevance of the newly identified RBM in Mph1 for BIR. The deleted sequence may contain another functional motif between aa 953 and 982. Even the Mph1 peptide used in the RPA binding assays (Figure 4D) starts from aa971, suggesting that there was no need for such an extended deletion. Therefore, a much shorter truncation (1-982) or a cluster of aa substitution in RBM should be used in the genetic experiments to address the importance of RBM in Mph1.\nAuthors’ response: In the revised version of MS we validate the mph1_1-954 mutant by comparison with a new mutant which harbours a single amino acid change: Phe993Ala. Our AlphaFold modelling shows that Mph1 Phe993 binds into the well-defined ‘side-pocket’ on Rfa1-NTD and equivalent hydrophobic residue-side-pocket interactions strongly enhance the affinity of many RPA clients for Rfa1 (Wu Y et al., Elife 12:e81639 (2023) doi: 10.7554/eLife.81639). Consistent with our interpretation that mph1_1-954 cells exhibit enhanced BIR efficiency due to disruption of the newly defined Mph1 C-terminal RBM, mph1-F933A cells exhibit a comparable increase in BIR efficiency. We conclude that classic RBM-meditated RPA-binding is required for the antagonistic role of Mph1 in BIR. This experiment is referred to in the revised version of the MS on page 13, lines 330-335:\n“(…), we mutated Mph1 Phe993 to alanine and found that this single amino acid change results in significantly elevated BIR efficiency, phenocopying Mph1_1-954 (Fig. 4E). We conclude that the high-affinity RPA binding mode which relies on RBM contacts within the Rfa1 NTD basic-hydrophobic groove and the hydrophobic ‘side-pocket’ (ref. 14) is critical for Mph1 to engage and disassemble D-loop intermediates during BIR.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "In the revision, the authors present additional data examining the contribution of the phospho-activated RPA-binding module (RBM) of Pol32 to break-induced replication (BIR). While these data support the idea that Pol32 can interact with RPA and the RBM contributes to BIR, it remains unclear whether phospho-activated RBM-RPA interaction plays a meaningful role in regulating BIR efficiency.\nAlthough the pol32-RBM mutation diminishes the RPA–Pol32 interaction, it does not measurably affect BIR on its own. If the RPA-binding module (RBM) contributes specifically to BIR through RPA binding, then a mutation in the RBM would be expected to cause a corresponding specific defect. However, an effect of the RBM mutation on BIR efficiency is observed only in Pol32 variants lacking the C-terminal PCNA-binding and Pol alpha–binding domains. These findings suggest that the RBM may contribute only redundantly to Pol32-dependent BIR, potentially independently of its interaction with RPA. As a result, it remains unclear whether the Pol32–RPA interaction plays a critical and specific role in BIR.\nMajor points\n1. The Pol32 RBM mutation significantly reduces the Pol31-RPA interaction (Fig. 2B); however, the RBM mutant does not exhibit a detectable defect in BIR (Fig. 2D). If the interaction with RPA plays a critical role in BIR function, a mutation in the RBM would be expected to cause a corresponding specific defect.\n2. The phosphomimetic Pol32-aspartate mutant exhibits increased BIR efficiency compared with wild-type cells (Fig. 3F). However, the corresponding alanine substitution (phospho-null) mutant behaves similarly to wild type (Fig. 3F). These results indicate that phosphorylation at these sites is dispensable for BIR efficiency. It therefore remains possible that the aspartate substitutions enhance Pol32 function through effects unrelated to phosphorylation, rather than faithfully mimicking a phosphorylated state.\nOther points:\n1. The in vitro kinase assays presented in Fig. 3 do not identify the specific kinase(s) responsible for phosphorylation of these residues. The data are also consistent with the possibility that RBM phosphorylation is mediated by a single, as-yet-unidentified kinase that was not tested in these assays. Therefore, the current data do not necessarily provide evidence that multiple kinases phosphorylate the RBM domain of Pol32.\n2. The Pol32 RBM mutation significantly reduces the Pol31–RPA interaction (Fig. 2B). The authors could address whether the phosphomimetic pol32-aspartate mutation enhances the RPA–Pol31 interaction, or the pol32-alanine mutation decreases this interaction.\n3. Structural models suggest that the RBM is positioned in close proximity to PCNA and Pol31 when Pol δ is bound to the PCNA–DNA complex (Fig. 5A). However, Pol3 and Pol31 (shaded) or ssDNA-bound RPA are not fully incorporated into the current structural model, it remains uncertain whether the RBM of Pol32 is spatially accessible for interaction with RPA once Pol δ is fully engaged with PCNA on DNA (Fig. 5D). Incorporating improved or alternative structural models, along with an expanded discussion, would strengthen the proposed mechanism by which Pol32–RPA interactions contribute to BIR efficiency.\nTechnical points:\nThere are no appropriate negative controls in Fig. 2B and supporting raw data. In particular, the POL32 strain should contain TAP-tagged POL31 in addition to POL32-FLAG. In addition, there is no validation shown for the anti-Rfa1 antibody. At least, IP–Western and input (extract) Western samples should be run on the same gel and probed with the anti-Rfa1 antibody to demonstrate specificity and reliability.\nRPA and Pol δ are localized to the lagging strand during DNA replication. Therefore, it remains possible that RPA and Pol δ are co-immunoprecipitated indirectly through ongoing lagging-strand DNA synthesis. To confirm a direct interaction between Rfa1 and Pol32, cell extracts used for co-IP experiments should be prepared from G1- or G2/M-arrested cells, where DNA replication is absent.", "reviewer_2": "The revised manuscript is improved, but a few points still need to be addressed.\n(Page 7, lines 181–184) The manuscript states that Pol32 RBM* shows reduced BIR kinetics at 8 h (Fig. 2E), but the data do not clearly support this description. The difference does not appear significant, as the error bars overlap at the 6 h and/or 8 h time points.\nMore broadly, Pol32 RBM* alone does not show even a weak BIR phenotype in cells. Only when combined with elimination of the Pol32 PIP box does the RBM mutation appear to have an effect. Because the phenotypes supporting the importance of the Pol32–RPA interaction are weak, any statement in the abstract implying a major regulatory function for Pol32–RPA should be substantially toned down. The Discussion could also include some speculation as to why Pol32 RBM* phenotypes are observed only in mutants that also eliminate the PIP box.\nDespite these remaining comments, this reviewer finds the revised work very interesting, as it demonstrates that multiple proteins important for BIR interact with RPA, and that these interactions can either stimulate or inhibit the process.\nMinor comments:\nLine 100: Should this be “~4-fold,” based on the data in the figure?\nQuestion #3 regarding the levels of crossover in the mph1 mutant was not addressed.", "reviewer_3": "I appreciate all the effort the authors put into addressing the comments on the original submission and the resubmitted manuscript did improve by adding more clarity to the conclusions through providing additional data. Unfortunately, adding more experiments haven't changed the overall message. The newly identified RBM in Pol32 has very little if any relevance to the efficiency of BIR in the context of full length Pol32. This is supported by multiple pieces of data on mutating the RBM in different ways - deleting it completely (figure 1), replacing a couple critical residues with Rs (RBM* mutant, figure 2) or mutating the phosphorylatable threonines into As or Ds (Figure 3). I appreciate that all the in vitro analyses show that the phosphorylation of these threonines improves the Pol32-RPA interaction, yet there is no effect in vivo, unless the Pol32-PCNA interaction is lost. For this reason, I find the title and the second part of the statement in the abstract \"Phosphorylation of the Pol32 RBM at Thr256 and Thr257 increases its affinity for Rfa1, while corresponding phospho-mimetic amino-acid substitutions promote BIR efficiency in vivo\" misleading as they don't mention the context of broken Pol32-PCNA interaction needed to observe the effect relevant to the Pol32-RPA interaction. One can't deny that the RBM is real and that the reported phosphorylation might be critical for some process involving the Pol32-RPA interaction, but the authors' own experiments prove that BIR is not this process because the Pol32-PCNA interaction is sufficient for the fully efficient BIR.\nInvestigating the RBM in Pif1 deeper also ruled out the importance of its RBM phosphorylation for BIR in vivo, even though the phosphorylation of S70 and S72 has been previously reported and as the authors discovered, it does increase the RPA binding in vitro.\nThe newly identified RBM in Mph1 also looks real based on the modelling and in vivo analysis, even though the authors didn't analyse the relevance of the suggested phosphorylation on S989 and S991 by mutagenesis to address its possible role in either RPA binding or in BIR directly (in vivo).\nThe model presented in Figure 4F is interesting but not justified by the experiments presented in the paper. The phosphorylation of Pol32 RBM is supposed to play a critical molecular role in it regulating the BIR progression, yet the loss of this RBM has no effect on BIR efficiency in vivo. The model in Figure 5D is more appropriate for the manuscript and justified by the presented discoveries."}, "author_response": "NCOMMS-25-05463A\n“Break-induced replication is enhanced by a phospho-activated RPA-binding module in Pol32“\nPoint-by-point Response to Referees\nReviewer #1 (Remarks to the Author):\nIn the revision, the authors present additional data examining the contribution of the phospho-activated RPA-binding module (RBM) of Pol32 to break-induced replication (BIR). While these data support the idea that Pol32 can interact with RPA and the RBM contributes to BIR, it remains unclear whether phospho-activated RBM-RPA interaction plays a meaningful role in regulating BIR efficiency.\nAlthough the pol32-RBM mutation diminishes the RPA–Pol32 interaction, it does not measurably affect BIR on its own. If the RPA-binding module (RBM) contributes specifically to BIR through RPA binding, then a mutation in the RBM would be expected to cause a corresponding specific defect. However, an effect of the RBM mutation on BIR efficiency is observed only in Pol32 variants lacking the C-terminal PCNA-binding and Pol alpha–binding domains. These findings suggest that the RBM may contribute only redundantly to Pol32-dependent BIR, potentially independently of its interaction with RPA. As a result, it remains unclear whether the Pol32–RPA interaction plays a critical and specific role in BIR.\nMajor points\n1. The Pol32 RBM mutation significantly reduces the Pol31-RPA interaction (Fig. 2B); however, the RBM mutant does not exhibit a detectable defect in BIR (Fig. 2D). If the interaction with RPA plays a critical role in BIR function, a mutation in the RBM would be expected to cause a corresponding specific defect.\nAuthors’ response: Our data presented in Fig. 1E, Fig. 2D, Fig. 2E, and Supplementary Fig. S1F demonstrate the BIR efficiency defect associated with loss of the Pol32 PIP-box is severely exacerbated upon disruption of the novel Pol32 RBM. This shows that in absence of the Pol32 PIP-box, BIR efficiency is dependent on the Pol32 RBM and thus that Pol32-RPA interactions facilitate successful D-loop DNA synthesis across the 30 kb or 100kb-long chromosomal regions that need to be replicated for a viable BIR outcome in the respective tester strains. A similar effect is observed if we mutate the phosphorylation sites Thr256 and Thr257 within the Pol32 RBM to alanine, which results in significant drop in BIR efficiency in pol32_1-133_T256A T257A cells as compared to pol32_1-133 cells (please see Fig. 3G). Thus, the availability of phosphorylation sites at Pol32 Thr256 and Thr257, which are phosphorylated in vivo (refs. 40-44 in our MS), result in enhanced D-loop DNA synthesis. Consistently, phospho-mimicking amino acid changes Pol32 T256D and T257D result in significantly enhanced BIR efficiency in pol32_1-133_T256D T257D cells as compared to pol32_1-133 cells (Fig. 3G).\nImportantly, full-length Pol32 T256D T257D-expressing cells (i.e., in the presence of the PIP-box) exhibit BIR efficiency levels that are significantly above wild-type levels (Fig. 3F), phenocopying cells in which the novel RBM in BIR antagonist Mph1 is disrupted (Fig. 4E). We also show that phosphorylation of the Pol32 RBM increases the affinity to RPA more than160-fold, which we rationalize by structural modelling showing that phosphorylation enables a high-affinity binding conformation of the Pol32 RBM on Rfa1 (please compare Fig. 2A and Fig. 3D). These data show that high-affinity RPA interaction by BIR agonist Pol32 and BIR antagonist Mph1 enhance or diminish BIR efficiency, respectively, strongly suggesting that interactions between the BIR machinery and the Rfa1 basic-hydrophobic groove are utilized to orchestrate D-loop DNA synthesis and disassembly at RPA-bound BIR intermediates.\nThese molecular insights are not diminished by the observation that disruption of the Pol32 RBM alone does not result in an overt BIR efficiency defect in HO-based BIR genetic assays. However, we realize that a detailed discussion on this point was lacking and, as also requested by Referee 2, we have now included a mechanistic model for the different functional contributions of the Pol32 PIP-box and RBM to BIR in the Discussion section on page 15, line 406 to page 16, line 424:\n“Disruption of the Pol32 RBM is associated with a slight delay in BIR kinetics, but, unlike removal of the PCNA-binding PIP-box, does not translate into an overt BIR efficiency defect in our reporter strains unless the PIP-box is also removed. PCNA encircles the DNA and acts as a processivity factor for Pol d. Our structural models of the Pol d-PCNA-DNA complex indicate that the Pol32 PIP-box binds PCNA, thus providing a physical connection between the processivity clamp and Pol d at the primer junction. This is consistent with biochemical results33 demonstrating that the Pol32 PIP-box enhances DNA synthesis by Pol d-PCNA in vitro. The Pol32 RBM provides physical interactions outside the core BIR replisome, with RPA-bound ssDNA that exists in the immediate vicinity of the D-loop primer-junction. We envisage that such interactions affect Pol d processivity less directly by promoting the recruitment of Pol d to D-loops and/or aiding its retention if Pol d disengages prematurely from PCNA. This model is consistent with the ability of the Pol32 RBM to compensate to some extent for the loss of the PIP-box, when BIR efficiency becomes profoundly dependent on the RBM and the Thr256/257 phosphorylation sites needed to enhance the interaction strength with RPA. While the functional contribution of the RBM to BIR efficiency is largely masked in HO-based experimental systems when Pol32-PCNA interactions are intact, it is revealed by enhanced BIR efficiency in the presence of Thr256/257 phospho-mimetic mutations, and we anticipate a particular relevance of the RBM for optimal Pol d recruitment/recruitment when BIR is in competition with alternative repair pathways at naturally occurring DSBs.”\n2. The phosphomimetic Pol32-aspartate mutant exhibits increased BIR efficiency compared with wild-type cells (Fig. 3F). However, the corresponding alanine substitution (phospho-null) mutant behaves similarly to wild type (Fig. 3F). These results indicate that phosphorylation at these sites is dispensable for BIR efficiency. It therefore remains possible that the aspartate substitutions enhance Pol32 function through effects unrelated to phosphorylation, rather than faithfully mimicking a phosphorylated state.\nAuthors’ response: In response to Specific Point 6 by Referee 1 in our first revision of the MS, we have demonstrated that the phospho-mimetic aspartate substitution at Pol32 Thr256 and Thr257 enhance the affinity for the Rif1 NTD in line with our data showing that phosphorylated Pol32 RBM peptides bind Rfa1 with much greater avidity than the non-phosphorylated form. Thus, the threonine to aspartate substitutions significantly increased the affinity of the Pol32 RBM for Rfa1 NTD, improving the K from 22.9 µM to 2.93 µM (please see Supplementary Fig. S6C). While it is always difficult to entirely rule out additional effects of any given mutation, our data are fully consistent with our interpretation that Pol32 T256D T257D enhances BIR efficiency (Fig. 3F) through increased affinity to RPA.\nOther points:\n1. The in vitro kinase assays presented in Fig. 3 do not identify the specific kinase(s) responsible for phosphorylation of these residues. The data are also consistent with the possibility that RBM phosphorylation is mediated by a single, as-yet-unidentified kinase that was not tested in these assays. Therefore, the current data do not necessarily provide evidence that multiple kinases phosphorylate the RBM domain of Pol32.\nAuthors’ response: Our data unequivocally demonstrate that the Pol32 RBM can be phosphorylated by multiple kinases as shown by kinase reactions with radioactive ATP (Supplementary Fig. S4) and mass spectrometry (Fig. 3A, B). Phosphorylation of the Pol32 RBM by PLK and DDK in vitro matches published data showing a dependence of Pol32 RBM phosphorylation on PLK and DDK which occurs in vivo (refs. 42 and 44 in our MS), whilst we found no evidence for direct phosphorylation by CDK (ref. 40). DDK, which showed the most robust activity towards the Pol32 RBM in our assays is particularly interesting as Cdc7 has been shown to promote BIR in vivo (ref. 46 in our MS). As we point out in the MS text, it is entirely possible that other kinases can phosphorylate the Pol32 RBM; indeed, we show that CKII (like Cdc7 an acidophilic kinase) can phosphorylate the Pol32 RBM; yet in vivo, only CDK, PLK, and DDK have been experimentally implicated thus far (ref. 40-44). We acknowledge the possibility of further complexities in Pol32 RBM phosphorylation in the MS text on page 17, line 453ff: “However, the details of Pol32 phospho-status regulation, which may involve additional kinases as well as phosphatases, remain to be elucidated in future studies”.\n2. The Pol32 RBM mutation significantly reduces the Pol31–RPA interaction (Fig. 2B). The authors could address whether the phosphomimetic pol32-aspartate mutation enhances the RPA–Pol31 interaction, or the pol32-alanine mutation decreases this interaction.\nAuthors’ response: We address enhanced Pol32-RPA interactions upon phosphorylation of the Pol32 RBM directly by quantitative biophysical FP experiments using purified components, showing that phosphorylation at Pol32 residues Thr256 and Thr257 increases the affinity for Rfa1 ~160-fold as compared to the non-phosphorylated Pol32 RBM. These data are robust and conclusive and, in our view, would not be strengthened by further IP experiments using Pol32 with surrogate phospho-mimetic amino acid changes in the Pol32 RBM.\n3. Structural models suggest that the RBM is positioned in close proximity to PCNA and Pol31 when Pol δ is bound to the PCNA–DNA complex (Fig. 5A). However, Pol3 and Pol31 (shaded) or ssDNA-bound RPA are not fully incorporated into the current structural model, it remains uncertain whether the RBM of Pol32 is spatially accessible for interaction with RPA once Pol δ is fully engaged with PCNA on DNA (Fig. 5D). Incorporating improved or alternative structural models, along with an expanded discussion, would strengthen the proposed mechanism by which Pol32–RPA interactions contribute to BIR efficiency.\nAuthors’ response: In revision 1 of our MS (in response to Specific Point 1 of Referee 1), we have greatly extended our structural analyses of the Pol d-PCNA-DNA complex (Fig. 5A, B and Supplementary Fig. S9A). This modelling comprises Pol d in its entirety (i.e., including Pol3 and Pol31, as stated in the MS on page 13, line 344f.) bound with PCNA on DNA. The results consistently show that the Pol32 RBM is accessible for the Rfa1 NTD when Pol d is engaged with PCNA on DNA (Fig. 5A and Supplementary Fig. S9A); to highlight this key point, which is the most relevant to the current MS, and aid the reader in their interpretation of a rather complex three-dimensional object, Pol3 and Pol31 are shown as ‘shaded outlines’ in Fig. 5A (this is stated in the accompanying figure legend on page 34, line 1000 of the MS: “For clarity, molecular cartoons for Pol31 and Pol3 are omitted, but with their relative positions represented by shaded polygons”). We therefore believe that the aspect of the Pol32-RBM being capable of interacting with the Rfa1-NTD, whilst Pol d is fully engaged with PCNA on DNA – through simultaneous engagement of the Pol3 CysA/PIP and Pol32-PIP motifs – is comprehensively addressed within the MS text. Whilst we share the desire of Referee 1 for even more complex modelling including RPA on ssDNA-containing recombination intermediates, it is our opinion that AlphaFold, in its current iteration, cannot deliver a single, globally correct architecture for the Pol d-PCNA-DNA complex engaged with RPA on a D-loop structure. This is in part due to the long length and structural complexity of a D-loop substrate, and also the lack of sequence-specificity for any of the modelled components that could serve as ‘molecular anchors’ to provide a defined orientation (a point that is especially true for the RPA trimer that can bind to ssDNA in multiple registers and modes). However, as pointed out in our MS, considering the conformational flexibility afforded by the long linker sequence between the Rfa1 NTD and subsequent RPA subdomains, and the flexibility of the disordered Pol32 C-terminal domain, it is highly plausible that Pol32 within the Pol d-PCNA-DNA complex could interact with RPA bound to a displaced ssDNA strand at a D-loop, as illustrated in our working models shown in Fig. 5C and D.\nWe have followed the recommendation to expand the Discussion section in order to address the mechanism by which we envisage Pol32-RPA interactions contribute to BIR efficiency more directly as detailed in our response to Major point 1 above.\nTechnical points:\nThere are no appropriate negative controls in Fig. 2B and supporting raw data. In particular, the POL32 strain should contain TAP-tagged POL31 in addition to POL32-FLAG. In addition, there is no validation shown for the anti-Rfa1 antibody. At least, IP–Western and input (extract) Western samples should be run on the same gel and probed with the anti-Rfa1 antibody to demonstrate specificity and reliability.\nRPA and Pol δ are localized to the lagging strand during DNA replication. Therefore, it remains possible that RPA and Pol δ are co-immunoprecipitated indirectly through ongoing lagging-strand DNA synthesis. To confirm a direct interaction between Rfa1 and Pol32, cell extracts used for co-IP experiments should be prepared from G1- or G2/M-arrested cells, where DNA replication is absent.\nAuthors’ response:\n(1) The requested control strain harbouring Pol31-TAP in addition to Pol32-FLAG is the experimental strain shown in the respective middle lanes of the WBs for cell extracts and TAP-IP in Fig. 2B. This may not have been entirely clear from the labelling of the figure, and we have re-labelled the WBs shown in Fig. 2B in the revised version of the MS to improve clarity.\n(2) The Source Data file has been updated to include the raw data for the immunoblot quantifications for Fig. 2B in the revised version of the MS.\n(3) The anti-Rfa1 antibody that we are using, Agrisera AS21 4551, is identical to Abcam ab221198. This antibody has been validated by Abcam and is widely used in the literature (for a recent publication by the Diffley, Aguilera, Gómez-González labs using the antibody, please see Núñez-Martín I et al., Nucleic Acids Res 53:gkaf707 (2025) doi: 10.1093/nar/gkaf707). To be clear that the anti-Rfa1 antibody is a tried and tested reagent, we have added the Abcam ab221198 specification to the Reporting summary for the revised version of the MS.\n(4) As described in the Methods section, all IP experiments are conducted in the presence of Pierce Universal Nuclease, which completely digests any DNA and RNA in the extracts, eliminating the possibility of DNA-mediated Pol d–RPA interactions in our IPs. Furthermore, we show that the Pol32 RBM binds Rfa1 in a direct protein-protein interaction in an in vitro system with purified components (please see Fig. 2C and Fig. 3C), which is in line with the severe reduction of Pol d-RPA co-immunoprecipitation to background levels when the Pol32 RBM is removed from the Pol d (Fig. 2B), a result that would not be expected if DNA-mediated interactions were involved.\nReviewer #2 (Remarks to the Author):\nThe revised manuscript is improved, but a few points still need to be addressed.\n(Page 7, lines 181–184) The manuscript states that Pol32 RBM* shows reduced BIR kinetics at 8 h (Fig. 2E), but the data do not clearly support this description. The difference does not appear significant, as the error bars overlap at the 6 h and/or 8 h time points.\nMore broadly, Pol32 RBM* alone does not show even a weak BIR phenotype in cells. Only when combined with elimination of the Pol32 PIP box does the RBM mutation appear to have an effect. Because the phenotypes supporting the importance of the Pol32–RPA interaction are weak, any statement in the abstract implying a major regulatory function for Pol32–RPA should be substantially toned down. The Discussion could also include some speculation as to why Pol32 RBM* phenotypes are observed only in mutants that also eliminate the PIP box.\nAuthors’ response: We thank Referee 2 for their insightful comments. While we observe a slight delay in early BIR kinetics upon mutation of the Pol32 RBM (Fig. 2E), we agree that the contribution of the Pol32 RBM to BIR efficiency is only overtly apparent upon additional mutation of the Pol32 PIP-box. In contrast, phospho-mimetic Pol32 RBM mutations enhance BIR efficiency both in the presence or absence of the Pol32 PIP-box (Fig. 3F and G). We have therefore followed the recommendation of Referee 2 to tone down the regulatory implications of Pol32 RBM phosphorylation, instead emphasising the phospho-dependent enhancement of RPA interaction and BIR efficiency observed for full-length Pol32. Accordingly, we have changed the title of the MS from “Break-induced replication is regulated by a phospho-activated RPA-binding module in Pol32” to “Break-induced replication is enhanced by a phospho-activated RPA-binding module in Pol32”. In the Abstract, we have changed the sentence reading “These results suggest that Pol32 functions as a rheostat regulating BIR efficiency by fine-tuning the affinity of Pol d for BIR intermediates that are bound by RPA” to “These results suggest that Pol32 functions as a rheostat where phosphorylation enhances the affinity of Pol d for BIR intermediates bound by RPA, thereby boosting BIR efficiency” in the revised version of the MS.\nSecondly, we now highlight the distinction between physical interactions provided by the Pol32 PIP-box, which strengthen the association of Pol d and its processivity clamp PCNA, and those provided by the Pol32 RBM with RPA-bound ssDNA around the D-loop structure. The former are of immediate relevance to processive DNA synthesis, the latter promote processivity only indirectly by supporting the assembly and retention of the BIR replisome at D-loops, which we propose provides a plausible mechanistic explanation for the apparent dominant role of the Pol32 PIP-box in facilitating BIR outcomes in HO-based BIR reporter assays. We apologize for not being more explicit on this point in the previous version of the MS and have added the following paragraph to the Discussion section to lay out our interpretation of the distinct functional roles of the Pol32 RBM and PIP-box (page 15, line 406 to page 16, line 424):\n“Disruption of the Pol32 RBM is associated with a slight delay in BIR kinetics, but, unlike removal of the PCNA-binding PIP-box, does not translate into an overt BIR efficiency defect in our reporter strains unless the PIP-box is also removed. PCNA encircles the DNA and acts as a processivity factor for Pol d. Our structural models of the Pol d-PCNA-DNA complex indicate that the Pol32 PIP-box binds PCNA, thus providing a physical connection between the processivity clamp and Pol d at the primer junction. This is consistent with biochemical results33 demonstrating that the Pol32 PIP-box enhances DNA synthesis by Pol d-PCNA in vitro. The Pol32 RBM provides physical interactions outside the core BIR replisome, with RPA-bound ssDNA that exists in the immediate vicinity of the D-loop primer-junction. We envisage that such interactions affect Pol d processivity less directly by promoting the recruitment of Pol d to D-loops and/or aiding its retention if Pol d disengages prematurely from PCNA. This model is consistent with the ability of the Pol32 RBM to compensate to some extent for the loss of the PIP-box, when BIR efficiency becomes profoundly dependent on the RBM and the Thr256/257 phosphorylation sites needed to enhance the interaction strength with RPA. While the functional contribution of the RBM to BIR efficiency is largely masked in HO-based experimental systems when Pol32-PCNA interactions are intact, it is revealed by enhanced BIR efficiency in the presence of Thr256/257 phospho-mimetic mutations, and we anticipate a particular relevance of the RBM for optimal Pol d recruitment/recruitment when BIR is in competition with alternative repair pathways at naturally occurring DSBs.”\nDespite these remaining comments, this reviewer finds the revised work very interesting, as it demonstrates that multiple proteins important for BIR interact with RPA, and that these interactions can either stimulate or inhibit the process.\nMinor comments:\nLine 100: Should this be “~4-fold,” based on the data in the figure?\nAuthors’ response: We thank Referee 2 for pointing this out and have changed the MS accordingly.\nQuestion #3 regarding the levels of crossover in the mph1 mutant was not addressed.\nAuthors’ response: This pertains to a previous question of whether an Mph1 C-terminal mutant is associated with higher crossover frequencies during DSB repair. Crossover frequency measurements require extended or unlimited homology on either side of a DSB, while BIR is measured between limited-homology substrates; thus, the genetic recombination assays deployed in this study do not report on crossover frequency. However, the ability of Mph1 to supress crossovers (instead promoting synthesis-dependent strand annealing at DSBs) and to restrict BIR outcomes are equally underpinned by Mph1-dependent dissociation of the invading DNA strand from D-loops (Prakash R et al., Genes Dev 23:67-79 (2009) doi: 10.1101/gad.1737809). Our results from revision 1 of the MS showing that disrupting the Mph1 RBM phenocopies MPH1 loss for elevated BIR efficiency and enhances D-loop stability in a PCR-based BIR assay provide very strong evidence that Mph1-dependent D-loop dissociation relies on Mph1-RPA interactions. While this predicts crossover frequency will be equally affected upon disruption of the Mph1 C-terminal RBM (with less effective D-loop disruption resulting in pathway choice away from synthesis-dependent stand annealing to more crossover outcomes), we feel that an expansion into other DSB repair pathways more peripherally linked to the BIR focus of our MS is not essential to draw the conclusion that the Mph1 RBM promotes the BIR antagonistic, well-established D-loop dissociation function of Mph1.\nReviewer #3 (Remarks to the Author):\nI appreciate all the effort the authors put into addressing the comments on the original submission and the resubmitted manuscript did improve by adding more clarity to the conclusions through providing additional data. Unfortunately, adding more experiments haven't changed the overall message. The newly identified RBM in Pol32 has very little if any relevance to the efficiency of BIR in the context of full length Pol32. This is supported by multiple pieces of data on mutating the RBM in different ways - deleting it completely (figure 1), replacing a couple critical residues with Rs (RBM* mutant, figure 2) or mutating the phosphorylatable threonines into As or Ds (Figure 3). I appreciate that all the in vitro analyses show that the phosphorylation of these threonines improves the Pol32-RPA interaction, yet there is no effect in vivo, unless the Pol32-PCNA interaction is lost. For this reason, I find the title and the second part of the statement in the abstract \"Phosphorylation of the Pol32 RBM at Thr256 and Thr257 increases its affinity for Rfa1, while corresponding phospho-mimetic amino-acid substitutions promote BIR efficiency in vivo\" misleading as they don't mention the context of broken Pol32-PCNA interaction needed to observe the effect relevant to the Pol32-RPA interaction. One can't deny that the RBM is real and that the reported phosphorylation might be critical for some process involving the Pol32-RPA interaction, but the authors' own experiments prove that BIR is not this process because the Pol32-PCNA interaction is sufficient for the fully efficient BIR.\nAuthors’ response: Referee 3 is right in pointing out that while the mutation of the Pol32 RBM results in a slight delay in BIR kinetics (Fig. 2E), this does not cause an overt BIR efficiency phenotype in our HO-based BIR report systems. However, it is important to consider that the contribution of the Pol32 RBM to BIR efficiency is unmasked upon removal of the Pol32 PIP-box, a condition under which BIR efficiency is dependent on the Pol32 RBM, which shows that Pol32-RPA interactions facilitate the completion of BIR along extended chromosomal regions (30 kb to 100kb) (Fig. 1E, Fig. 2D, Fig. 2E, and Supplementary Fig. S1F). Similarly, mutation of the Thr256/257 phosphorylation sites in Pol32 RBM largely abolishes BIR outcomes in pol32_1-133_T256A T257A cells (Fig. 3G). This is compatible with different functional roles which we envisage for the Pol32 RBM vs. the PIP-box modules in promoting BIR: the Pol32 PIP-box directly connects Pol d to its processivity clamp PCNA, thereby supporting continuous BIR DNA synthesis; the Pol32 RBM links Pol d to RPA-bound ssDNA within D-loops, thus only indirectly supporting processivity by promoting BIR replisome recruitment and/or retention at D-loops. This mechanistic difference provides a conceptual model to explain why the presence of the Pol32 PIP-box largely masks the contribution of the newly identified Pol32 RBM to promoting BIR outcomes in HO-based experimental systems. We realize that we were not clear enough on this point and now elaborate on the potential cellular function of the Pol32 RBM for BIR in an extended Discussion section in the revised version of the MS (page 15, line 406 to page 16, line 424):\n“Disruption of the Pol32 RBM is associated with a slight delay in BIR kinetics, but, unlike removal of the PCNA-binding PIP-box, does not translate into an overt BIR efficiency defect in our reporter strains unless the PIP-box is also removed. PCNA encircles the DNA and acts as a processivity factor for Pol d. Our structural models of the Pol d-PCNA-DNA complex indicate that the Pol32 PIP-box binds PCNA, thus providing a physical connection between the processivity clamp and Pol d at the primer junction. This is consistent with biochemical results33 demonstrating that the Pol32 PIP-box enhances DNA synthesis by Pol d-PCNA in vitro. The Pol32 RBM provides physical interactions outside the core BIR replisome, with RPA-bound ssDNA that exists in the immediate vicinity of the D-loop primer-junction. We envisage that such interactions affect Pol d processivity less directly by promoting the recruitment of Pol d to D-loops and/or aiding its retention if Pol d disengages prematurely from PCNA. This model is consistent with the ability of the Pol32 RBM to compensate to some extent for the loss of the PIP-box, when BIR efficiency becomes profoundly dependent on the RBM and the Thr256/257 phosphorylation sites needed to enhance the interaction strength with RPA. While the functional contribution of the RBM to BIR efficiency is largely masked in HO-based experimental systems when Pol32-PCNA interactions are intact, it is revealed by enhanced BIR efficiency in the presence of Thr256/257 phospho-mimetic mutations, and we anticipate a particular relevance of the RBM for optimal Pol d recruitment/recruitment when BIR is in competition with alternative repair pathways at naturally occurring DSBs.”\nWith respect to the statement (Abstract) “Phosphorylation of the Pol32 RBM at Thr256 and Thr257 increases its affinity for Rfa1, while corresponding phospho-mimetic amino-acid substitutions promote BIR efficiency in vivo.”, this is fully supported by the data presented in our MS (please see Fig. 3F showing that full-length Pol32 T256D T257D-expressing cells exhibit BIR efficiency levels that are significantly above wild-type levels). Reflecting the phosphorylation-dependent enhancement of Pol32 RBM-RPA interaction and the boost in BIR efficiency observed for Pol32 T256D T257D (in the presence of the PIP-box), and following a recommendation by Referee 2, we have changed the title of the MS from “Break-induced replication is regulated by a phospho-activated RPA-binding module in Pol32” to “Break-induced replication is enhanced by a phospho-activated RPA-binding module in Pol32”.\nInvestigating the RBM in Pif1 deeper also ruled out the importance of its RBM phosphorylation for BIR in vivo, even though the phosphorylation of S70 and S72 has been previously reported and as the authors discovered, it does increase the RPA binding in vitro.\nAuthors’ response: Please note that phosphorylation of Pif1 Ser70 and Ser72 results in an approximate 2-fold increase of an already high affinity of the Pif1 RBM for RPA (Supplementary Fig. S8). This is in stark contrast to an approximate 160-fold increase in affinity for the Pol32 RBM (which binds Rfa1 with a moderate affinity in the non-phosphorylated form) upon phosphorylation at Thr256 and Thr265. As we explain in the MS (please refer to section “Pol32 pRBM, Pif1, and Mph1 similarly target Rfa1” on page 11), this can be rationalized by the presence of a negative charge within Pif1 (Asp71) equivalent to Thr256 in Pol32 that allows Pif1 to engage with Rfa1 in the ‘high-affinity’ mode with Pif1 Trp75 projecting into the Rfa1 hydrophobic ‘side-pocket’ independently of phosphorylation. In contrast, Pol32 RBM phosphorylation is required for ‘side-pocket’ interactions of Pol32 Leu262. Consistently, cells expressing (full-length) Pol32 T256D T257D in vivo exhibit a boost in BIR efficiency (Fig. 3F) while Pif1 S70D S72D expression is no different from Pif1 wild-type expression in terms of BIR efficiency. This provides strong evidence that Pol32 RBM phosphorylation does promote BIR, and we argue that this is not thrown into question, but rather corroborated by the finding that Pif1 RBM phosphorylation is not required for high-affinity RPA binding nor BIR efficiency.\nThe newly identified RBM in Mph1 also looks real based on the modelling and in vivo analysis, even though the authors didn't analyse the relevance of the suggested phosphorylation on S989 and S991 by mutagenesis to address its possible role in either RPA binding or in BIR directly (in vivo).\nAuthors’ response: As explained in our response to the preceding point, our data show that RBMs exist in two flavours and either support phosphorylation-dependent or constitutive high-affinity Rfa1 binding. Like Pif1, Mph1 falls into the latter category (Mph1 Asp990 has equivalency with Pif1 Asp71 allowing high-affinity binding in the non-phosphorylated form, please refer to Fig. 4A, D in our MS), for which our analysis of Pif1 serves as an exemplar case.\nThe model presented in Figure 4F is interesting but not justified by the experiments presented in the paper. The phosphorylation of Pol32 RBM is supposed to play a critical molecular role in it regulating the BIR progression, yet the loss of this RBM has no effect on BIR efficiency in vivo. The model in Figure 5D is more appropriate for the manuscript and justified by the presented discoveries.\nAuthors’ response: As detailed in our response to Referee 3’s first point above, we now provide an extended discussion to explain why the removal of the PIP-box is required to unmask the contribution of the Pol32 RBM to BIR efficiency. With regard to the model Fig. 4F, we disagree that this is not supported by the data. We show in Fig. 3F that phospho-mimicking mutations T256D T257D within full-length Pol32 are associated with a significant increase in BIR efficiency over wild-type Pol32, which is fully consistent with the depiction in Fig. 4F. Upon request, we can relegate Fig. 4 panel F to the supplementary information, but we feel this would be a loss as this illustration serves as a visual summary of results for a broader audience."}]

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