# RAD51 protects abasic sites to prevent replication fork breakage

Yodhara Wijesekara Hanthi, Miguel Angel Ramirez-Otero, Robert Appleby, Anna de Antoni, Luay Joudeh, Vincenzo Sannino, Salli Waked, Alessandra Ardizzoia, Viviana Barra, Daniele Fachinetti, et al.

<figure><img src="image_1.png" /></figure>

► To cite this version:

Yodhara Wijesekara Hanthi, Miguel Angel Ramirez-Otero, Robert Appleby, Anna de Antoni, Luay Joudeh, et al.. RAD51 protects abasic sites to prevent replication fork breakage. Molecular Cell, 2024, 84 (16), pp.3026-3043.e11. 10.1016/j.molcel.2024.07.004. hal-04774245

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<figure><img src="image_2.png" /></figure>
# Molecular Cell

## RAD51 protects abasic sites to prevent replication fork breakage

### Graphical abstract

<figure><img src="image_3.png" /></figure>

### Authors

Yodhara Wijesekara Hanthi,
Miguel Angel Ramirez-Otero,
Robert Appleby, ..., Daniele Fachinetti,
Luca Pellegrini, Vincenzo Costanzo

### Correspondence

lp112@cam.ac.uk (L.P.),
vincenzo.costanzo@ifom.eu (V.C.)

### In brief

Hanthi et al. found that RAD51 recognizes and binds to abasic sites. Together with BRCA2, RAD51 prevents the accumulation of abasic site-induced replicative DNA gaps (caused by DNA methylation, oxidation, and deamination) and averts replication fork breakage, resulting from MRE11-dependent abasic site cleavage within single-stranded DNA, thus ensuring genomic stability.

### Highlights

* Cryo-EM structure shows that RAD51 specifically recognizes and binds to abasic sites

* RAD51 prevents MRE11-dependent abasic DNA cleavage, suppressing fork breakage

* BRCA2 and RAD51 also restrict the accumulation of abasic sites and replicative gaps

* RAD51 protects abasic sites resulting from DNA methylation, oxidation, and deamination

<figure><img src="image_4.png" /></figure>
## Article

# RAD51 protects abasic sites to prevent replication fork breakage

Yodhara Wijesekara Hanthi,<fnref n="1" />,<fnref n="5" /> Miguel Angel Ramirez-Otero,<fnref n="1" />,<fnref n="5" /> Robert Appleby,<fnref n="3" />,<fnref n="5" /> Anna De Antoni,<fnref n="1" /> Luay Joudeh,<fnref n="3" /> Vincenzo Sannino,<fnref n="1" /> Salli Waked,<fnref n="1" /> Alessandra Ardizzoa,<fnref n="1" /> Viviana Barra,<fnref n="4" /> Daniele Fachinetti,<fnref n="4" /> Luca Pellegrini,<fnref n="3" />,* and Vincenzo Costanzo<fnref n="1" />,<fnref n="2" />,<fnref n="6" />,*

## SUMMARY

Abasic sites are DNA lesions repaired by base excision repair. Cleavage of unrepaired abasic sites in single-stranded DNA (ssDNA) can lead to chromosomal breakage during DNA replication. How rupture of abasic DNA is prevented remains poorly understood. Here, using cryoelectron microscopy (cryo-EM), Xenopus laevis egg extracts, and human cells, we show that RAD51 nucleofilaments specifically recognize and protect abasic sites, which increase RAD51 association rate to DNA. In the absence of BRCA2 or RAD51, abasic sites accumulate as a result of DNA base methylation, oxidation, and deamination, inducing abasic ssDNA gaps that make replicating DNA fibers sensitive to APE1. RAD51 assembled on abasic DNA prevents abasic site cleavage by the MRE11-RAD50 complex, suppressing replication fork breakage triggered by an excess of abasic sites or POLθ polymerase inhibition. Our study highlights the critical role of BRCA2 and RAD51 in safeguarding against unrepaired abasic sites in DNA templates stemming from base alterations, ensuring genomic stability.

## INTRODUCTION

Abasic sites, referred to as apurinic and apyrimidinic (AP) sites, are among the most frequent DNA lesions.<fnref n="1" /> AP sites are predominantly repaired by base excision repair (BER) via AP endonuclease (APE1)-mediated DNA strand incision and subsequent gap filling within double-stranded (ds)DNA.<fnref n="2" /> AP sites can arise through spontaneous hydrolysis of the base-sugar bond and following the removal of altered bases by BER glycosylases.

Considerable amounts of AP sites can be generated by demethylation of 5-methyl-2'-deoxycytidine (5mdC) through teneleven translocation (TET) methylcytosine dioxygenases that create intermediates like 5-hydroxy-methyl-2'-deoxycytidine (5hmdC), 5-formyl-2'-deoxycytidine (5fdC), and 5-carboxyl-2'-deoxycytidine (5cadC), which thymine DNA glycosylase (TDG) can remove when these are paired with guanine.<fnref n="3" /> Single-strand selective monofunctional uracil DNA glycosylase (SMUG1) can instead remove uracil or 5-hydroxymethyl-uracil (5hmU) from single-stranded (ss) in addition to dsDNA,<fnref n="4" />,<fnref n="5" /> possibly arising from base deamination and/or oxidation,<fnref n="6" /> forming AP sites<fnref n="3" /> that must be carefully processed to avoid replication fork damage.

Unrepaired DNA base damage and AP sites confer toxicity to homologous recombination (HR)-defective cells.⁷⁻¹³

DNA synthesis arrest and restart at DNA polymerase stalling lesions results in ssDNA gaps trailing behind replication forks in the absence of HR factors RAD51 and BRCA1/2.¹³⁻²¹

SMUG1 plays a major role in ssDNA gap formation in the absence of functional HR proteins.¹³ However, the mechanisms responsible for the accumulation of ssDNA gaps during DNA replication in the absence of HR proteins remain elusive. Furthermore, it is yet to be determined if HR proteins have a direct influence on AP sites, apart from their established role in repairing double-strand breaks (DSBs) that might result from the processing of these lesions. Addressing these questions is challenging due to the essential role of key HR factor RAD51.

Here, we employed a multidisciplinary approach including cryoelectron microscopy (cryo-EM) imaging and reconstruction of RAD51 nucleoprotein filaments (NPFs), biochemical assays with purified proteins, Xenopus laevis (Xl) egg extracts, and DNA EM to study the mechanisms underlying HR factors-mediated control of AP-site metabolism.

We also developed a new method to identify AP sites within ssDNA gaps occurring at replication forks. This technique is

<footnote n="1">¹IFOM, The AIRC Institute of Molecular Oncology, Milan, Italy</footnote>

<footnote n="2">²Department of Oncology and Hematology-Oncology, University of Milan, Milan, Italy</footnote>

<footnote n="3">³Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK</footnote>

<footnote n="4">⁴Institute Curie, PSL Research University, CNRS, UMR 144, 26 Rue d'Ulm, 75005 Paris, France</footnote>

<footnote n="5">⁵These authors contributed equally</footnote>

<footnote n="6">⁶Lead contact</footnote>

<footnote>*Correspondence: lp112@cam.ac.uk (L.P.), vincenzo costanzo@ifom.eu (V.C.)</footnote>

<footnote>https://doi.org/10.1016/j.molcel.2024.07.004</footnote>
useful to highlight the presence of spontaneous AP sites in repli-
cating templates isolated from HR-defective cancer cells.

Our results demonstrate that RAD51 can directly recognize AP
sites by exploiting its natural binding mechanism to DNA and that
the presence of AP sites increases RAD51's association rate
with DNA, protecting AP sites from uncontrolled processing
and suppressing their accumulation. These functions of RAD51
are critical in preventing replication fork breakage caused by
excessive AP sites, especially when compensatory mecha-
nisms, including POL0 polymerase-dependent abasic ssDNA
gap filling, are impaired.

Remarkably, the accumulation of abasic ssDNA gaps and fork
breakage can be suppressed by inhibiting TET2 oxidase,
APOBEC3B deaminase, or DNA methyltransferase 1 (DNMT1),
highlighting enzymatic base modifications as a major source of
AP sites that lead to the accumulation of ssDNA gaps in HR-
defective vertebrate cells.

RESULTS

Structural basis for the recognition of abasic DNA
by RAD51

In the cryo-EM structure of the RAD51 NPFs, the phospho-
ribose backbone of DNA is embedded within a protein sheath
of helically arranged RAD51 molecules, whereas the bases
project out toward the solvent.²² The DNA-binding loops L1
and L2 of RAD51 grip the phospho-ribose backbone, unstack-
ing the bases of filament DNA into triplets of nucleotides that
remain in approximate B-DNA conformation. Given its mode
of DNA interaction, we reasoned that RAD51 binding to abasic
DNA should be possible or even more favorable compared with
normal DNA. Thus, the gap in the DNA caused by the missing
base would constitute a preferential site of filament nucleation,
as the bases flanking the AP site are already unstacked. Elec-
trophoretic mobility shift assays indicated that RAD51 can form
NPFs with ss- and ds-DNA containing abasic DNA sites with
equivalent apparent affinity as with normal DNA (Figures S1A
and S1B). Furthermore, kinetic analysis of binding using sur-
face plasmon resonance showed that the rate of RAD51 asso-
ciation with DNA oligonucleotides increases in proportion to the
number of abasic sites present in the sequence (Figures 1A and
S1C). Biochemical evidence supported a model whereby
RAD51 would preferentially bind to the AP site with its DNA-
binding loops L1 and L2 rather than unstacking the bases of
adjacent nucleotides. We tested this hypothesis by cryo-EM
structure determination of a RAD51 NPF containing a ssDNA
oligonucleotide with multiple abasic sites positioned 3-nt apart
to match the known footprint of RAD51 on DNA.²² We
reasoned that if RAD51 did bind preferentially to the regularly
spaced AP sites in the DNA sequence, the filament structure
determined by helical averaging would show no density for
the base of the nucleotide recognized by loops L1 and L2 of
each RAD51 protomer. Alternatively, if RAD51 bound indiffer-
ently to abasic or normal sites, helical averaging would yield
the same density for the base of all nucleotides in the filament.

The 3.2 Å cryo-EM structure of a RAD51 NPF bound to a
23-nt oligonucleotide with 7 regularly spaced AP sites showed
the complete absence of density for the base of the nucleotide

engaged by RAD51 loops L1 and L2 (Figures 1B, 1C, S2A, and S2B; Table 1). Thus, the experiment proved that the AP sites had determined the position of the RAD51 protomers on filament DNA, in support of a preference for RAD51 binding to AP sites. In the structure, RAD51 binds abasic ssDNA in a similar way as observed for the structure of the pre-synaptic filament,²³ except that the N-terminal segment of loop L2 has shifted to partially occupy the gap of the missing base with the side chain of V273 (Figures 1D and 1E).

AP sites can occur on both ss- and ds-DNA. We sought to
determine if RAD51 can recognize AP sites in the context of
dsDNA, and whether it showed preference for the DNA strand
carrying AP sites within a DNA double helix. We reconstituted
a RAD51 NPF on dsDNA using the same oligonucleotide car-
rying regularly spaced AP sites that had been annealed to its
complementary sequence with no abasic sites. To stabilize
short dsDNA, we reduced the number of AP sites to the cen-
tral five, so that each end would have 5-nt long tracts of
normal dsDNA. Cryo-EM structure determination of this fila-
ment by helical averaging at 3.6 Å showed strongly reduced
density for the base at the nucleotide position of the inner
strand recognized by RAD51 loops L1 and L2, whereas the
outer strand of dsDNA in the filament showed density for a
full complement of bases (Figures 2A, 2B, S3A, and S3B;
Table 1). Overall, RAD51 interacts with the abasic dsDNA in
a highly similar mode as in the post-synaptic filament
(Figures 2C and 2D).²³

This experiment demonstrated the same mode of RAD51
binding to AP sites in dsDNA observed for abasic ssDNA. It
further showed that RAD51 can discriminate between abasic
and normal DNA strands within the double helix, by preferentially
binding the abasic strand.

AP sites stimulate RAD51 chromatin recruitment

To validate RAD51's binding to AP sites, we analyzed RAD51
chromatin association in XI egg extract following APE1
inhibition using AR03²⁶ and APE1 inhibitor III (APE1i).²⁷,²⁸
These treatments increased RAD51 chromatin binding without
causing DNA DSBs, as evidenced by the absence significant
levels of phosphorylated histone H2AX (γH2AX) in the treated
samples (Figures 3A and 3B) and the lack of ataxia telangiecta-
sia and Rad3-related (ATR) chromatin enrichment normally
induced by stalled and broken replication forks.¹⁵ The presence
of AP sites was confirmed using a biotinylated aldehyde reac-
tive probe (ARP),²⁹ which detected more AP sites in nuclei
treated with APE1i (Figures 3C and 3D). Control experiments
with sperm nuclei treated with non-saturating amounts of
methyl methane sulfonate (MMS)¹⁴ validated the ARP's detec-
tion of AP sites (Figures 3C and 3D). A complementary enzyme-
linked immunosorbent assay (ELISA) conducted on genomic
DNA isolated from treated extracts corroborated AP-site induc-
tion (Figure 3E).

To validate the effects of APE1 inhibition on RAD51 chromatin
recruitment, we produced XI SMUG1 (xSMUG1) (Figure S4A), a
DNA glycosylase that removes uracil from ssDNA, creating AP
sites.<sup>4,5,30</sup> xSMUG1's activity was confirmed *in vitro* with active
recombinant XI APE1 (xAPE1) (Figure S4B), which was capable
<figure><img src="image_5.png" /></figure>

of cleaving an AP site-containing oligo (Figure S4C) and an uracil-containing oligo exposed to xSMUG1 (Figure S4D).

An excess xSMUG1 enhanced RAD51's chromatin binding, exceeding the levels induced by aphidicolin (APH)¹⁵ or SMUG1/APH combination, indicating a rise in AP site formation akin to APE1 inhibition (Figures 3F and 3G).

RAD51 discriminates abasic strands within dsDNA (Figures 2A-2D). Using geminin to halt DNA replication and unwind- ing,³¹ we observed that xSMUG1-generated AP sites in dsDNA also enhanced RAD51's chromatin binding, suggesting that AP sites promote RAD51 recruitment beyond ssDNA contexts (Figures 3H and 3I).

<caption>Figure 1. RAD51 binds to ssDNA containing abasic sites</caption>

(A) Surface plasmon resonance analysis of RAD51's interaction with abasic ssDNA. The biotinylated DNA ligand was coupled to a streptavidin-coated sensor chip, and the RAD51 analyte was added to the mobile phase to measure the RU response. The left panel shows representative sensorgrams of RAD51 binding to DNA oligonucleotides with 1, 3, and 5 AP sites, and no AP sites as control. The right panel reports the apparent association rates ($K_{on}$) of RAD51 to the abasic DNA oligonucleotides, from sensogram data analysis.

(B) Cryo-EM structure of a RAD51 nucleoprotein filament with ssDNA containing multiple, periodically spaced AP sites (AP7). The RAD51 protomers in the filament are drawn as ribbons and the ssDNA is in spacefill representation.

(C) Cryo-EM density of the ssDNA in the filament, with fitted oligonucleotide structure. The abasic nucleotides are colored pink and their position is indicated by an arrow. The DNA sequence is shown next to the structure; abasic site positions are marked by x.

(D) RAD51's DNA-binding loops L1 and L2 interaction with the AP site. Loops are drawn as light blue ribbons, while the DNA is in light-brown filled-ring representation with the abasic nucleotide colored pink.

(E) Superposition of pre-synaptic RAD51 NPF (PDB: 8BQ2) with the NPF on abasic ssDNA (this work). Two rotated views of the filament are shown, centered on the position of the AP site. See also Figures S1 and S2.

## RAD51-dependent protection of abasic DNA from nuclease-mediated cleavage

To evaluate AP sites' impact on replicating genomes, we examined DNA replication forks isolated from APE1i or SMUG1-treated extracts using EM.¹⁴,¹⁵,³² These agents induced ssDNA gaps, increasing the total length of ssDNA accumulating at replication forks (Figures 4A, 4B, and S4E). Inhibiting RAD51 DNA binding with GST-BRC4

peptide (BRC4) led to larger ssDNA gaps on intact forks, possibly from MRE11 exonuclease activity on nascent DNA¹⁴,¹⁵,³² (Figures 4B, 4C, S4F, and S4G).

Remarkably, APE1i or SMUG1 treatment with BRC4 led to breaks in nearly 50% of forks, as shown by the presence of asymmetric branches, indicating severing of one of the replicated arms¹⁵ (Figures 4D, 4E, S4F, and S4G). Frequent breaks on gapped strands (Figures 4D, S4F, and S4G) suggest that ssDNA gaps evolved into DSBs.¹⁵

To determine the fork breakage mechanism, we examined nucleases present at forks known to cut AP sites. The human MRE11-RAD50 (MR) complex³³ could cleave AP sites from
<table><caption>**Table 1.** Cryo-EM data collection and real-space refinement</caption><thead><tr><th colspan="3">Data collection</th></tr><tr><th></th><th>Abasic ssDNA NPF (EMDB: EMD-19050, PDB: 8RCD)</th><th>Abasic dsDNA NPF (EMDB: EMD-19051, PDB: 8RCF)</th></tr></thead><tbody><tr><td>Microscope</td><td>Titan Krios G3</td><td></td></tr><tr><td>Voltage (keV)</td><td>300</td><td></td></tr><tr><td>Detector</td><td>Gatan K3</td><td></td></tr><tr><td>Collection mode</td><td>counting</td><td></td></tr><tr><td>Magnification</td><td>130,000x</td><td></td></tr><tr><td>Defocus range (µm)</td><td>-2.6, -2.4, -2.2, -2.0, -1.8, -1.6, -1.4, -1.2, -1.0, -0.8</td><td></td></tr><tr><td>No. movies</td><td>10,623</td><td>10,084</td></tr><tr><td>Frames/movie</td><td>49</td><td>44</td></tr><tr><td>Pixel size (Å/pixel)</td><td>0.652</td><td></td></tr><tr><td>Electron dose (e<sup>-</sup>/Å<sup>2</sup>/s)</td><td>41.5</td><td>44.14</td></tr><tr><td>Exposure (s)</td><td>1.33</td><td>1.20</td></tr><tr><td>Picked particles</td><td>851,378</td><td>1,791,680</td></tr><tr><td>Final particles</td><td>183,769</td><td>8853</td></tr><tr><td>Processing method</td><td>helical reconstruction</td><td></td></tr><tr><td>asm units (#)</td><td>6</td><td></td></tr><tr><td>Helical twist (°)</td><td>56.3</td><td>56.0</td></tr><tr><td>Helical rise (Å)</td><td>16.2</td><td>15.8</td></tr><tr><th colspan="3">Resolution estimates (Å)</th></tr><tr><th></th><th>Masked</th><th>Unmasked</th><th>Masked</th><th>Unmasked</th></tr><tr><td>d<sub>FSC</sub> (half maps; 0.143)</td><td>3.2</td><td>3.4</td><td>3.6</td><td>3.8</td></tr><tr><td>d<sub>gg</sub> (full/half1/half2)</td><td>3.1/4.2/4.2</td><td>3.0/3.7/3.7</td><td>3.6/2.7/2.7</td><td>3.6/2.7/2.7</td></tr><tr><td>d<sub>model</sub></td><td>3.2</td><td>3.2</td><td>3.5</td><td>3.5</td></tr><tr><td>d<sub>FSC model</sub> (0/0.143/0.5)</td><td>2.9/3.0/3.3</td><td>2.9/3.0/3.5</td><td>3.2/3.3/3.6</td><td>3.2/3.4/3.8</td></tr><tr><th colspan="5">Real-space refinement</th></tr><tr><th colspan="3">Composition</th></tr><tr><td>Chains</td><td>9</td><td></td><td>10</td><td></td></tr><tr><td>Atoms</td><td>19,680 (hydrogens: 0)</td><td></td><td>20,202 (hydrogens: 0)</td><td></td></tr><tr><td>Residues</td><td>protein: 2,480 nucleotide: 23</td><td></td><td>protein: 2,488 nucleotide: 46</td><td></td></tr><tr><td>Water</td><td>0</td><td></td><td>0</td><td></td></tr><tr><td>Ligands</td><td>Ca: 16, ATP: 8</td><td></td><td>Ca: 16, ATP: 8</td><td></td></tr><tr><th colspan="5">Model</th></tr><tr><th colspan="3">Bonds (RMSD)</th></tr><tr><td>Length (Å) (# > 4σ)</td><td>0.009 (0)</td><td></td><td>0.008 (2)</td><td></td></tr><tr><td>Angles (°) (# > 4σ)</td><td>0.615 (0)</td><td></td><td>0.629 (0)</td><td></td></tr><tr><td>MolProbity score<sup>24</sup></td><td>2.04</td><td></td><td>1.74</td><td></td></tr><tr><td>Clash score</td><td>11.65</td><td></td><td>9.12</td><td></td></tr><tr><th colspan="5">Ramachandran plot (%)</th></tr><tr><td>Outliers</td><td>0.0</td><td></td><td>0.0</td><td></td></tr><tr><td>Allowed</td><td>7.19</td><td></td><td>1.47</td><td></td></tr><tr><td>Favored</td><td>92.81</td><td></td><td>98.53</td><td></td></tr><tr><td>Rotamer outliers (%)</td><td>0.05</td><td></td><td>2.16</td><td></td></tr><tr><td>Cbeta outliers (%)</td><td>0.0</td><td></td><td>0.0</td><td></td></tr><tr><th colspan="5">ADP (B factors)</th></tr><tr><td>Iso/Aniso (#)</td><td>19,680/0</td><td></td><td>20,202/0</td><td></td></tr></tbody></table>

(Continued on next page)
<table><caption>**Table 1. Continued**</caption><thead><tr><th colspan="3">Data collection</th></tr><tr><th></th><th>Abasic ssDNA NPF (EMDB: EMD-19050, PDB: 8RCD)</th><th>Abasic dsDNA NPF (EMDB: EMD-19051, PDB: 8RCF)</th></tr></thead><tbody><tr><td colspan="3">min/max/mean</td></tr><tr><td>Protein</td><td>25.79/122.15/65.94</td><td>62.75/129.35/91.93</td></tr><tr><td>Nucleotide</td><td>55.91/63.38/58.35</td><td>40.27/193.54/75.20</td></tr><tr><td>Ligand</td><td>47.74/64.34/55.28</td><td>50.09/86.04/82.91</td></tr><tr><td colspan="3">Model vs. data</td></tr><tr><td>CC mask<sup>25</sup></td><td>0.85</td><td>0.77</td></tr><tr><td>CC box</td><td>0.58</td><td>0.58</td></tr><tr><td>CC peaks</td><td>0.52</td><td>0.45</td></tr><tr><td>CC volume</td><td>0.83</td><td>0.76</td></tr><tr><td>Mean CC for ligands</td><td>0.87</td><td>0.83</td></tr></tbody></table>

UDG-treated ssDNA oligos,³⁴ matching human APE1 efficiency (Figure S4H).

The MRE11 endonuclease inhibitor PFM01³⁵ reduced broken forks induced by AP-site accumulation in the absence of RAD51 bound to DNA, implicating MRE11 in cleaving abasic ssDNA gaps (Figures 4E and 4F). PFM01 also reduced fork breakage induced by SMUG1 excess without RAD51 (Figures 4E and S4I). RAD51 inhibited both MR complex and APE1-dependent cleavage of AP site-containing oligos (Figures 4G, 4H, and S4J), suggesting that RAD51 NPFs block nuclease access to DNA.

As POLθ can bypass AP sites³⁶ and fill ssDNA gaps,¹⁵,²¹ we verified abasic gap sealing by POLθ. Adding a 2.5-fold excess of the full length of X/ POLθ¹⁵ to egg extract reduced ssDNA gaps and fork breakage induced by APE1i or xSMUG1 treatment in the absence of RAD51 bound to DNA (Figures 4B and 4E).

Overall, these findings suggest that RAD51 binding and POLθ-mediated DNA synthesis across AP sites prevent abasic gaps conversion to DSBs.

## AP sites trigger ssDNA gaps in APEX1- or BRCA2-defective cells

To verify whether AP sites are directly linked to spontaneous ssDNA gaps that occur during DNA replication in the absence of RAD51 bound to DNA, we set up a novel assay based on replicating DNA fibers analysis coupled with APE1 nuclease treatment (Figures 5A and 5B). Unlike S1 nuclease,³⁷ APE1 shortens DNA fibers only when encountering AP sites containing ssDNA (Figure 5A), allowing the detection of AP sites in replicating DNA templates.

To conduct the APE1-fiber assay, replication forks were sequentially labeled with chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU) base analogs. Permeabilized nuclei were then exposed to APE1 or S1 nucleases (Figure 5B), which were equally able to shorten IdU tracts on DNA fibers isolated from APE1i-exposed DLD1 wild-type (WT) cells, indicating the presence of AP site-containing ssDNA gaps (Figure 5B). Noticeably, APE1i reduced fork speed as shown by shorter IdU track lengths even without enzyme treatment, consistent with AP sites' ability to decrease fork progression³⁸ (Figure 5B).

Genome-wide rise in AP sites from APE1i was confirmed with AA3, a cell-permeable alkoxyamine that tags endogenous AP sites that can be fluorescently labeled via click chemistry for AP-site quantification on either nascent or parental DNA.³⁹ AA3 fluorescence increased with APE1i concentration, confirming AP-site accumulation (Figures S5A and S5B). Nuclear fluorescence, concentrated in areas potentially more prone to AP sites formation,⁴⁰ was evident even at low APE1i doses (Figure S5B).

To validate the results obtained with APE1i, we used CRISPR-Cas9 to generate APEX1⁻/⁻ DLD1 cells (Figure S5C), which showed increased unrepaired AP sites, as measured by ELISA and AA3 fluorescence (Figures 5C–5E). As expected, APE1i did not increase toxicity in APEX1⁻/⁻ cells any further, unlike in BRCA2⁻/⁻ cells, which were APE1i sensitive (Figure S5D).

IdU tracks on fibers from APEX1⁻/⁻ cells were shorter after APE1 or S1 nuclease treatment, confirming the formation of abasic ssDNA gaps in the absence of APE1 activity (Figure 5F).

Importantly, both BRCA2⁻/⁻ cells and those with silenced BRCA2 or RAD51 showed IdU-labeled fiber shortening following APE1 treatment (Figures 5F, S5E, and S5F). This effect was absent in CldU-labeled segments replicated earlier (Figure S5G). These results suggest that AP sites within ssDNA gaps arise in the proximity of active replication forks in the absence of RAD51 or BRCA2 and are sealed as replication forks progress.

SMUG1 knockdown, confirmed by immunoblot (Figure S5H), suppressed abasic ssDNA gaps in APEX1⁻/⁻ and BRCA2⁻/⁻ DLD1 cells (Figure 5G) and APE1i-treated WT cells (Figure S5I), indicating a key role for SMUG1 in abasic ssDNA gap formation. SMUG1 knockdown also slowed fork progression in cells with intact APEX1 (Figures 5G and S5I), likely due to the accumulation of uncleared modified bases, a phenomenon that remains to be further defined.

## BRCA2- and RAD51-mediated suppression of AP sites

Surprisingly, AP sites were more prevalent in BRCA2⁻/⁻ than WT or APEX1⁻/⁻ cells (Figures 5C–5E and S5J) and in X/ nuclei with compromised RAD51 chromatin binding (Figures S5K and S5L), indicating that DNA-bound RAD51 may inhibit AP-site accumulation.
<figure><img src="image_6.png" /></figure>

Accordingly, uracil-containing ssDNA preincubated with RAD51 resisted AP-site formation by xSMUG1, as shown by the lack of xAPE1-mediated cleavage following RAD51 and SMUG1 inactivation (Figure 5H). This suggests RAD51 shielding of DNA from glycosylase action.

SMUG1 downregulation reduced AP-site accumulation in asynchronously growing BRCA2⁻/⁻ cells but did not completely suppress it (Figure S5J). This is possibly due to additional processes contributing to SMUG1-independent AP formation in the absence of RAD51 bound to non-replicating dsDNA. However, complete AP-site suppression occurred in APEX1⁻/⁻ cells, suggesting effective control of an intact BRCA2/RAD51 function over AP sites' accumulation (Figure S5J).

Considering POL0's ability to bypass AP sites,³⁶ we explored its role in gap suppression. Inhibiting POL0 polymerase with POL0i, a previously characterized inhibitor,¹⁵,²¹ increased abasic ssDNA gap formation in APEX1⁻/⁻ and BRCA2⁻/⁻ cells, indicating that

<caption>**Figure 2. RAD51 binds to the abasic strand in dsDNA**</caption>

(A) Cryo-EM structure of a RAD51 nucleoprotein filament containing abasic ssDNA (AP5) annealed to its complementary DNA sequence (APc).

(B) Cryo-EM density of the dsDNA in the filament, with fitted dsDNA structure. Abasic nucleotides are colored in pink. Their position is indicated by an arrow. Inset on the right shows a superposition of cryo-EM maps for abasic dsDNA (this work) in gray and dsDNA in the post-synaptic RAD51 filament²³ in transparent yellow. The weak density still visible at the AP site position is the result of helical averaging including normal nucleotides at the two outermost positions in the AP5-APc dsDNA, so that a residual signal corresponding to 2/7 of normal base density would be expected for exclusive recognition of AP sites by RAD51.

(C) Interaction of RAD51's DNA-binding loops L1 and L2 with the AP site in dsDNA. Loops are drawn as light blue ribbons. DNA is in filled-ring representation, colored light- and dark brown for the inner and outer DNA strands respectively, with the abasic nucleotide colored pink.

(D) Superposition of post-synaptic RAD51 NPF (PDB: 8BR2) with the NPF on abasic dsDNA (this work).

See also Figure S3.

POL0 significantly contributes to sealing abasic ssDNA gaps (Figure S5M).

Silencing of DNA-directed primase/polymerase protein (PRIMPOL) (Figures S6A and S6B), crucial for DNA synthesis restart and ssDNA gap formation without BRCA1/2,¹³,¹⁹,²⁰ significantly reduced abasic ssDNA gaps in APEX1⁻/⁻ and BRCA2⁻/⁻ cells and in those treated with APE1i (Figures S6C–S6F). PRIMPOL was required for normal fork progression in these contexts due to its AP-site bypass ability.

POL0i did not further increase AP-site gaps in PRIMPOL-independent APEX1⁻/⁻ cells, suggesting that POL0 fills gaps post-PRIMPOL synthesis (Figure S6C). Conversely, POL0i prevented full suppression of gaps mediated by PRIMPOL knockdown in BRCA2⁻/⁻ cells, indicating that POL0 fills PRIMPOL-independent abasic gaps arising in this context (Figure S6D), possibly on the lagging strand.¹⁵

To further verify the occurrence of SMUG1- and PRIMPOL-dependent abasic ssDNA gaps in BRCA2-deficient conditions, we used PEO1 cancer cells with a mutation producing a non-functional truncated BRCA2 (BRCA2Y1655X) and PEO1 reversion cells (C4-2) with restored BRCA2 (BRCA2W1)⁴¹,⁴² (Figure S6G). APE1- and S1-dependent DNA fiber shortening was observed only in mutant cells and was abolished by SMUG1 or PRIMPOL knockdown (Figure S6H), confirming abasic gap formation in these tumor cells.

Abasic site processing protein (HMCES) also protects DNA from AP sites during replication.²⁸ Accordingly, inhibition of
<figure><img src="image_7.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_8.png" /></figure>

RAD51 chromatin binding

ARP fluorescence

<figure><img src="image_9.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_15.png" /></figure>

(legend on next page)
HMCES expression (Figure S6I) increased abasic gaps in repli-
cating DNA isolated from WT DLD1 cells (Figure S6J). Instead,
in BRCA2⁻/⁻ cells, HMCES silencing reduced fork progression
and rendered DNA fibers resistant to S1 and APE1 treatment
(Figure S6J). This might be due to the increased recruitment of
slower translesion synthesis (TLS) polymerases filling abasic
gaps and reducing the fork rate, a process restricted by
HMCES.³⁸ The direct molecular relationship between HMCES
and TLS in BRCA2⁻/⁻ cells remains to be determined.

DNMT1-, APOBEC3B-, and TET2-dependent abasic DNA
gap accumulation

To investigate the origin of spontaneous abasic gaps, we explored
the possibility that AP sites originate from 5mdC turnover.

To this end, we targeted DNMT1 and DNMT3B DNA methyl-
transferases.⁴³ Using auxin-inducible degron (AID) technology,⁴⁴
we degraded DNMT1 alone (DNMT1<sup>NA</sup>) or in combination with
DNMT3B knockout (DNMT1<sup>NA</sup>/DNMT3B<sup>-/-</sup>) in DLD1 cells.⁴⁵
Treatment with indole-3 acetic acid (IAA) for 48 h resulted in
complete DNMT1 degradation in DNMT1<sup>NA</sup> (Figure S6K), leading
to a 50% reduction in 5mdC levels,⁴⁵ significantly restraining AP
site-containing ssDNA gaps in BRCA2-silenced cells (Figure 5I).
Similar results were obtained by inducing DNMT1 degradation in
DNMT3B<sup>-/-</sup> cells (Figures 5J and S6K), a combination leading to
a more pronounced 5mdC loss.⁴⁵ These results demonstrate a
direct connection between DNA methylation and abasic gap for-
mation in the absence of BRCA2. Accordingly, DNMT1 degrada-
tion significantly reduced the excess of total AP sites caused by
acute downregulation of BRCA2 in asynchronously growing cells
(Figures S6L and S6M).

5mdC is oxidized to 5hmdC by TET enzymes.³ TET2 knock-
down (Figure S7A) suppressed abasic ssDNA gap formation in
BRCA2⁻/⁻ cells (Figure 5K). Conversely, knockdown of TDG (Fig-
ure S7B), which removes 5fdC and 5cadC from dsDNA,³
reduced fork speed possibly due to the accumulation of un-
cleared modified bases in cells with intact APEX1 (Figure S7C).
However, it did not suppress ssDNA gaps in either APEX1⁻/⁻
or BRCA2⁻/⁻ DLD1 cells (Figure S7C), indicating that DNA de-
methylation occurring within dsDNA does not contribute to
ssDNA gap accumulation during replication.

The suppression of abasic ssDNA gaps by SMUG1 downregu-
lation, coupled with SMUG1's ability to excise 5hmU,⁴,⁵,³⁰ led us
to explore 5-hydroxymethyl-uridine (5hmdU) production mecha-
nisms in ssDNA.

We investigated APOBEC3B deaminase, which *in vitro* can convert 5hmdC to 5hmdU in ssDNA, albeit with low efficiency.⁴⁶ Silencing APOBEC3B, present in the nucleus of several cancer cells, including DLD1⁴⁷⁻⁵⁰ (Figure S7D), significantly reduced abasic ssDNA gap formation (Figure S7E). Additional APOBEC3 members expressed in DLD1 and other cancer cells, such as APOBEC3C, D, F, and H,⁴⁷⁻⁵⁰ may jointly contribute to the accumulation of abasic gaps.

MRE11-dependent cleavage of AP sites at replication
forks in BRCA2- and RAD51-defective cells

We then assessed AP sites' impact on replicating genomes in BRCA2⁻/⁻ cells. APE1i induced DSBs in BRCA2⁻/⁻ DLD1 cells, mostly in S-phase, as evidenced by γH2AX foci accumulation in EdU-positive and in a few non-S-phase cells (Figures 6A and 6B). MRE11 inhibition with PFM01 or MRE11 knockdown reduced γH2AX foci induced by APE1i in BRCA2⁻/⁻ and BRCA2-silenced APEX1⁻/⁻ cells (Figures 6A, 6B, and S7F-S7J). These results indicate that in human cells, an overload of AP sites induced by APE1 inhibition is not tolerated in the absence of RAD51 bound to DNA, similar to *Xl*.

DNA EM confirmed APE1i-induced breakage in replication
forks isolated from BRCA2<sup>-/-</sup> DLD1 cells, suppressed by
PFM01 (Figures 6C and 6D). These observations indicate that
BRCA2 and RAD51 protect against fork breakage induced by
AP site-cleaving nucleases and align with the known synthetic
lethality between APEX1 and BRCA2.<sup>3</sup>

We then tested POLθ's role in mitigating fork breakage. We
monitored γH2AX accumulation in WT and APEX1⁻/⁻ cells
exposed to POLθi following RAD51 or BRCA2 small-interfering
RNA (siRNA)-mediated knockdown (Figures 6E, 6F, S7K, and
S7L). POLθ polymerase inhibition further augmented γH2AX
foci formation triggered by defective APEX1, RAD51, or
BRCA2 (Figures 6E, 6F, S7M, and S7N). Consistent with these
findings, APEX1⁻/⁻ cells exhibited high sensitivity to POLθi (Fig-
ure S7O), although the effect was less pronounced compared

**Figure 3. RAD51 recruitment by AP sites containing chromatin**

(A) Representative immunoblot of chromatin assembled with sperm nuclei (4,000 n/µL), incubated in egg extract treated with DMSO, AR03 (10 µM), or APE1 inhibitor III (10 µM) and isolated at indicated times.

(B) Quantification of RAD51 chromatin binding as in (A) normalized to H2B. Horizontal bars indicate mean ± SD. Symbols indicate *n* = 3 independent biological replicate values. One-way ANOVA, followed by Dunnett's post hoc test for multiple comparisons; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001.

(C) Immunofluorescence of sperm nuclei treated with MMS (3 mg/mL) or APE1i, stained with DAPI and ARP, and detected with fluorescent avidin.

(D) Quantification of (C) showing individual nuclei intensity. Horizontal bars indicate mean ± SD of all data points. Overlay dots indicate independent *n* = 2 biological replicate medians. One-way ANOVA, followed by Dunnett's post hoc test; \* *p* < 0.05.

(E) ELISA quantification of AP sites in DNA from treated extracts. Horizontal bars indicate mean ± SD of all data points. Overlay dots indicate *n* = 4 independent biological replicate values. One-way ANOVA, followed by Dunnett's post hoc test; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.

(F) Immunoblot of chromatin derived from extracts treated with DMSO, APH (1.5 mM), recombinant xSMUG1 (0.5 µM), or both for the indicated times. His-tagged SMUG1 detected.

(G) Quantification of RAD51 chromatin binding as in (F) normalized to H2B. Horizontal bars indicate mean ± SD. Symbols indicate *n* = 3 independent biological replicate values. One-way ANOVA, followed by Dunnett's post hoc test; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001.

(H) Immunoblot of chromatin derived from extracts treated with buffer, xSMUG1, GEMININ (GEM) (25 ng/µL), or both for the indicated times.

(I) Quantification of RAD51 chromatin binding as in (H) normalized to H2B. Horizontal bars indicate mean ± SD. Symbols indicate *n* = 3 independent biological replicate values. One-way ANOVA, followed by Dunnett's post hoc test; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001.

See also Figure S4.
<figure><img src="image_16.png" /><figcaption>Figure 4. RAD51-mediated protection against MRE11-dependent abasic DNA processing and fork breakage</figcaption></figure>

(A) Representative EM image of a replication fork from egg extract treated with APE1i (10 µM, 45 min). Magnified area in rectangle. Cartoon dotted lines show ssDNA.

(B) Quantification of total ssDNA length for each fork isolated from extracts treated as indicated. Horizontal bars indicate the mean ± SD for a total of 72 forks. Overlay dots show *n* = 3 independent biological replicate means. One-way ANOVA, followed by Tukey's post hoc test for multiple comparisons; **p** < 0.01; ***p** < 0.001; ****p** < 0.0001.

(C and D) Representative EM images of forks from an extract treated with APE1i (10 µM) and GST-BRC4 (0.5 mg/mL, 45 min).

(E) Percentage of broken forks from extracts treated as indicated. Horizontal bars indicate mean ± SD relative to 72 forks. Overlay dots show *n* = 3 independent biological replicate means. One-way ANOVA, followed by Tukey's post hoc test; **p** < 0.01; ***p** < 0.001; ****p** < 0.0001.

(F) EM of a rescued fork from extract treated with APE1i, GST-BRC4, and PFM01 (100 µM). Base pair lengths numbered; gaps by red arrows; broken arm by black arrow.

(G) Denaturing gel showing fluorescein-labeled ssDNA with an AP-site generated by UDG-mediated uracil removal and treatment with buffer or 200 nM MR complex and increasing RAD51.

(H) AP oligo cleavage percentage in the presence of MR and increasing RAD51. No RAD51 sample was taken as reference. Horizontal bars indicate mean ± SD of all data points. Overlay dots show *n* = 3 independent replicates. One-way ANOVA, followed by Dunnett's post hoc test; ****p** < 0.0001; ns, non-significant.

See also Figure S4.

with BRCA2⁻/⁻ cells. These findings suggest BRCA2, RAD51, and POLθ counteract fork breakage from endogenous AP sites.

Remarkably, reducing DNA methylation prevented γH2AX foci increase induced by POLθi in BRCA2-silenced cells (Figures 6G and 6H), implying that base changes derived from methylated DNA promote DSB formation in the absence of BRCA2 and POLθ polymerase activity.

DISCUSSION

RAD51 plays an essential role in higher eukaryotes due to its requirement for HR-based DSB repair.⁵¹⁻⁵³ However, its function in unchallenged DNA replication has also been increasingly

recognized. The absence of functional HR proteins results in the accumulation of ssDNA gaps during chromosomal DNA replication. This suggests additional roles for RAD51 that are independent of DSB repair.¹³⁻¹⁹ The origin and fate of these gaps remain unclear to date.

Our study shows that RAD51 suppresses ssDNA gaps' accumulation by regulating AP-site metabolism. We demonstrate that (1) RAD51 specifically recognizes AP sites, as shown through cryo-EM-mediated visualization of RAD51 NPFs; (2) the presence of AP sites increases RAD51's DNA association rate; (3) RAD51 NPFs preferentially form on abasic DNA strands; (4) RAD51 NPFs hinder abasic DNA processing by AP nucleases, preventing replication fork breakage caused by MRE11-dependent cleavage of unrepaired AP sites; (5) APE1 treatment of DNA fibers reveals gaps containing AP sites in replicating DNA templates; (6) BRCA2 and RAD51 reduce AP site and abasic gaps' accumulation by limiting glycosylases access to DNA; (7) POLθ seals abasic gaps limiting DSB formation; and (8)
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

number of fibers: 363 217 497 221 202 224 203 203 208 464 249 525

<figure><img src="image_20.png" /></figure>

C

<figure><img src="image_21.png" /></figure>

D

<figure><img src="image_22.png" /></figure>

number of cells: 206 280 264

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

number of fibers: 373 204 203 375 203 204 204 203 203

<figure><img src="image_26.png" /></figure>

number of fibers: 454 198 255 400 110 321 297 203 173 389 273 256 390 208 401 411 302 387

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

number of fibers: 329 306 302 321 273 261 261 176 150 177 198 189

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

number of fibers: 291 168 239 367 210 294 204 376 261 291 292 184

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

number of fibers: 188 188 123 204 219 146

(legend on next page)
DNMT1, TET2, APOBEC3B, and SMUG1 contribute to abasic gaps formation and fork breakage.

Collectively, these results provide new insights into HR proteins' role in genome stability.

## Structural determinants of AP-site recognition

We demonstrate that RAD51 NPFs specifically recognize AP sites in both ss- and ds-DNA. This preference is related to RAD51's DNA-binding mechanism, wherein the L1 and L2 loops from its ATPase domain unstack bases into nucleotide triplets. An abasic nucleotide resembles a pre-formed RAD51 binding site, making the binding process energetically favorable. This is evidenced by the faster association with a DNA molecule containing even a single AP site. The ability to recognize AP sites suggests that these lesions are the likely preferred nucleation points for RAD51 polymerization on DNA.

Cryo-EM demonstrates RAD51's unique ability to selectively bind abasic DNA, assembling specifically on strands with AP sites in dsDNA. RAD51's selective binding to AP sites, independently of ssDNA gaps, is confirmed by increased recruitment to geminin-treated chromatin containing non-replicating abasic dsDNA.

The similarity in DNA recognition between RAD51 and its orthologs, including bacterial RecA,⁵³,⁵⁴ suggests that AP-site recognition might be a conserved trait, predating complex HR processes like strand pairing and homology searching. These results align with RecA's role in preventing DNA degradation by the UvrABC complex,⁵⁵ which can process DNA with AP sites.⁵⁶ As AP sites are a fundamental DNA vulnerability,¹ RAD51's protection of abasic DNA may represent a primordial function of this protein family.

## RAD51-mediated protection of abasic DNA from nucleases

RAD51's enhanced binding kinetics to abasic DNA is critical to protect AP sites from enzymatic processing possibly by outcom-

peting nucleases, thus preventing AP-site cleavage (Figure 7A). Among these, MRE11, a prominent S-phase nuclease,¹⁴,⁵⁷⁻⁵⁹ can cleave AP sites.³⁴ However, RAD51 effectively shields AP sites during replication, averting fork collapse. This protection persists until polymerases such as POLθ and possibly other TLS enzymes intervene to bypass the lesion and fill the gap induced by AP-site stalling nascent DNA synthesis (Figure 7A). POLθ's ability to displace RAD51 might facilitate this task.⁶⁰

EM analysis of replication intermediates with uneven fork branches¹⁵ shows that without RAD51, breakages mainly occur in areas with ssDNA gaps (Figure 7B).

Unlike RAD51, the RPA ssDNA binding protein complex may be less effective at preventing ssDNA cleavage by AP endonucleases. This is due to its dynamic binding to ssDNA⁶¹,⁶² that could briefly expose ssDNA to nucleases and glycosylases.⁶¹ In contrast, RAD51 stably encapsulates DNA within its NPF structure, shielding it from external access. A role for stable NPFs in protecting against different nucleases is consistent with BRCA2/RAD51's ability to suppress nascent DNA degradation at reversed replication forks⁶³ and ssDNA gaps,¹⁴ preventing extensive formation of ssDNA regions on replication intermediates.¹⁴,¹⁵,³²

Overall, our findings highlight the unique RAD51's function in safeguarding replicating DNA templates with AP sites from breaking. This role differs from the repair of DSBs possibly resulting from AP sites originating from nascent DNA misincorporation of BER substrates, including 5hmdU generated by cytoplasmic deaminases, that compromises the survival of cells with impaired DSB repair.¹¹,¹²

Considering APE1's preference for dsDNA⁶⁴ and our observations of increased γH2AX foci in APEX1⁻/⁻ cells after BRCA2 or RAD51 silencing, it is unlikely that APE1 significantly contributes to replication forks cleavage. However, RAD51 may prevent excessive nicking by APE1 at loci undergoing BER-mediated events leading to AP sites' formation in dsDNA ahead of replication forks, thus preventing DSBs occurrence at fork passage.

Figure 5. Replicative, abasic ssDNA gap accumulation in repair-defective human cancer cells

(A) Schematic of APE1 and S1 nuclease cleavage of abasic DNA causing DSBs.

(B) Scatter dot plot of IdU track lengths on the indicated number of DNA fibers from DLD1 WT cells treated with DMSO or APE1i, post S1 or APE1 treatment. Horizontal bars indicate mean ± SD of all data points from independent experiments. Overlay dots show *n* = 3 independent biological replicate means. Kruskal-Wallis test, followed by Dunn's post hoc test for multiple comparisons; ***p* < 0.01; **p* < 0.001; ***p* < 0.0001; ns, non-significant.

(C) ELISA-based AP-site quantification in DNA from WT, BRCA2⁻/⁻, or APEX1⁻/⁻ DLD1 cells. Horizontal bars indicate mean ± SD of all data points. Dots show *n* = 3 independent biological replicate means. One-way ANOVA, followed by Dunnett's post hoc test; *p* < 0.05.

(D) AP sites quantification in the indicated number of WT, BRCA2⁻/⁻, or APEX1⁻/⁻ DLD1 cells using Alexa Fluor 647-AA3. Bars indicate mean ± SD of all data points. Dots show *n* = 3 independent biological replicate medians. Boxes indicate interquartile with ranges. One-way ANOVA, followed by Dunnett's post hoc test; **p* < 0.01.

(E) Representative images of DLD1 WT, APEX1⁻/⁻, BRCA2⁻/⁻ nuclei with Alexa 647-AA3. Scale bars, 10 µm.

(F) IdU track lengths on the indicated numbers of DNA fibers from DLD1 WT, APEX1⁻/⁻, BRCA2⁻/⁻ cells, post buffer, S1, or APE1 treatment. Bars indicate mean ± SD of all data points. Overlay dots show *n* = 2 independent biological replicate medians. Kruskal-Wallis test, followed by Dunn's post hoc test; ****p* < 0.0001; ns, non-significant.

(G) Similar to (F), on fibers derived from cells treated with control or SMUG1 siRNAs. Bars indicate mean ± SD of all data points. Overlay dots show *n* = 3 independent biological replicate medians. Kruskal-Wallis test, followed by Dunn's post hoc test; ****p* < 0.0001; ***p* < 0.0001; ns, non-significant.

(H) Denaturing gel showing fluorescein-ssDNA with uracil incubated with RAD51, then xSMUG1, and boiled before xAPE1 treatment.

(I and J) (I) IdU track lengths on the indicated number of fibers from DNMT1<sup>NA</sup> and (J) DNMT1<sup>NA</sup>/DNMT3B⁻/⁻ DLD1 cells following treatment with control or BRCA2 siRNAs and DMSO or IAA. Cells were treated with S1 or APE1. Bars indicate mean ± SD of all data points. Overlay dots show *n* = 3 independent biological replicate medians. Kruskal-Wallis test, followed by Dunn's post hoc test; ****p* < 0.0001; ns, non-significant.

(K) IdU track lengths on the indicated number fibers from DLD1 BRCA2⁻/⁻ cells treated with control or TET2 siRNAs, post S1, or APE1 treatment. Bars indicate mean ± SD of all data points. Overlay dots show *n* = 3 independent biological replicate medians. Kruskal-Wallis, followed by Dunn's post hoc test; ****p* < 0.0001; ns, non-significant.

See also Figures S5–S7.
<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

APE1i + PFM01

BRCA2-/-

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

number of cells: 939 878 996 697

<caption>**Figure 6. BRCA2- and RAD51-mediated suppression of MRE11-mediated fork breakage**</caption>

(A) Images of γH2AX foci in EdU-labeled DLD1 WT and BRCA2⁻/⁻ cells post-treatment with DMSO, APE1i or APE1i and PFM01.

(B) Quantification of γH2AX foci in the indicated number of cells. Bars indicate mean ± SD of all data points. Overlay dots indicate n = 3 independent biological replicate means. One-way ANOVA, followed by Tukey's post hoc test for multiple comparisons; **p** < 0.01; ***p** < 0.001; ****p** < 0.0001; ns, non-significant.

(legend continued on next page)
## RAD51-mediated control of AP sites and abasic ssDNA gap accumulation

To study the impact of RAD51 on abasic DNA templates under-
going replication, we developed an assay based on APE1-
mediated cleavage of replicating DNA fibers. Abasic template
cleavage by APE1 leads to the shortening of the labeled
DNA fiber only when occurring in the context of a ssDNA
gap. This assay can therefore clearly distinguish AP sites pre-
sent in the replicating template from the ones occurring within
nascent DNA or un-replicated dsDNA ahead of the replica-
tion forks.

Using the APE1-fiber assay, we demonstrate that in the absence of BRCA2, which is required for the loading and the stabilization of RAD51 NPFs onto replicating DNA,<sup>65,66</sup> AP-site containing ssDNA gaps accumulate during DNA replication.

These results together with the overall AP increase measured in BRCA2-defective cells provide evidence that RAD51 not only shields AP sites from nuclease activity but also suppresses their accumulation. RAD51 trailing behind forks or nucleating at AP sites and spreading laterally could restrict the unfettered activity of enzymes that contribute to the formation of AP sites on exposed ssDNA strands. These enzymes include glycosylases and deaminases acting on ssDNA<sup>3-6,67</sup> (Figure 7C). This function could be important to prevent widespread base changes observed in HR-defective cells.<sup>68</sup> The similarity between Xl and human cells indicates RAD51's conserved role in managing AP sites across species.

The requirement for stable RAD51 NPFs to prevent DNA ac-
cess of AP sites inducing enzymes may explain ssDNA gap for-
mation in murine cells with the mouse equivalent of the
BRCA2<sup>S3291A</sup> mutation,<sup>69</sup> a base substitution that only minimally
affects human BRCA2-mediated stabilization of RAD51 NPFs, as
recently noted.<sup>66</sup> Efficient formation of NPFs nucleating from AP
sites might be required to prevent glycosylase access to adja-
cent ssDNA regions and restrain further abasic sites and gaps
accumulation.

The rapid delivery of RAD51 to abasic DNA necessary to outcompete cleavage by AP site-specific nucleases might be instead compromised by more severe BRCA2 mutations that also impact HR and cellular viability upon DNA damage.⁶⁹

The suppression of AP sites' accumulation by BRCA2/RAD51
might not be confined to ssDNA but could extend to dsDNA re-
gions, including enhancers, which preferentially bind RAD51<sup>70</sup>
and are subjected to TDG-dependent DNA demethylation,<sup>71</sup>
preventing further DNA damage.

## Compensatory mechanisms preventing deleterious consequences of AP sites' formation in the absence of BRCA2 or RAD51

In HR-defective cells, alternative pathways can partially
compensate for the absence of abasic DNA protection. One
such pathway relies on POLθ's ability to replicate across AP
sites,³⁶ by filling in the ssDNA gaps created by PRIMPOL reprim-
ing downstream of the AP lesion on the leading strand or arising
in between stalled Okazaki fragments, which would be normally
protected by BRCA2/RAD51.¹⁵ Abasic gap filling would
contribute to explain the synthetic lethality induced by POLθ
polymerase inhibitors in unchallenged BRCA2-defective cells
in addition to the established POLθ's role in DSB repair.¹⁵,²¹,⁷²

Additional protection is conferred by HMCES shielding AP sites,²⁸ and by TLS polymerases, which can also bypass AP sites,⁷³ serving as a backup pathway in HR-defective cells¹³,⁷⁴ or cells that overexpress APOBEC3 enzymes.³⁸,⁷⁵ The interplay among POLθ and TLS polymerases, including Pol ζ, is likely to be additive¹⁵,²¹ and highly regulated as already shown for HMCES, which restricts TLS polymerases engagement with replicating DNA.³⁸ The expression levels of these factors might impact on the number, the size and the fate of abasic ssDNA gaps.

## Origins of AP sites impacting DNA replication

Unexpectedly, DNMT1-dependent 5mdC production is a pre-
requisite for replicative abasic ssDNA gaps' accumulation and
DSBs' formation in the absence of BRCA2/RAD51- and POLθ-
mediated gap filling.

Physiological turnover of 5mdC is regulated by the TET en-
zymes, which oxidize 5mdC to 5hmdC and then 5fdC and
5cadC. These forms are removed by BER through TDG in the
context of dsDNA to replace modified cytosines with unmodified
ones³ (Figure 7D).

In contrast to TET2 downregulation, TDG silencing does not prevent abasic ssDNA gap formation in BRCA2<sup>-/-</sup> cells, suggesting that TDG-mediated 5mdC demethylation is not involved in replicative ssDNA gap formation, consistent TDG expression outside S-phase.³ However, we cannot exclude that a fraction of AP sites accumulating in the absence of BRCA2 is still contributed by TDG activity beyond S-phase.

Once generated, 5hmdC can be deaminated to yield 5hmdU, which is then processed by SMUG1 within ssDNA<sup>4,5,46</sup> (Figure 7C).

APOBEC3 and AID deaminases promote widespread cancer genome mutagenesis.⁷⁶ In particular, APOBEC3B, APOBEC3C, and APOBEC3D can be found expressed in multiple

<footnote>(C) EM of replication forks from BRCA2<sup>-/-</sup> DLD1 cells treated with DMSO, APE1i, or APE1i and PFM01.</footnote>

<footnote>(D) Percentage of broken forks from extracts treated as indicated. Bars indicate mean ± SD relative to a total of 72 forks. Overlay dots show *n* = 3 independent biological replicate means. One-way ANOVA, followed by Dunnet's post hoc test; ***** *p* < 0.0001.</footnote>

<footnote>(E and F) (E) Quantification of γH2AX foci in the indicated number of DLD1 cells treated with control, RAD51 or (F) BRCA2 siRNAs. Bars indicate mean ± SD of all data points. Overlay dots indicate *n* = 3 independent experiment means. One-way ANOVA, followed by Tukey's post hoc test; ** *p* < 0.01; *** *p* < 0.001; **** *p* < 0.0001; ns, non-significant.</footnote>

<footnote>(G) Images of γH2AX foci in EdU-labeled DNMT1<sup>NA</sup> DLD1 cells treated with BRCA2 siRNAs, followed by DMSO or 5 µM POLθi. Buffer or IAA were supplemented where indicated.</footnote>

<footnote>(H) Quantification of γH2AX foci shown in (G). Horizontal bars indicate mean ± SD of all data points. Overlay dots indicate independent biological replicate means. *n* = 3; one-way ANOVA, followed by Tukey's post hoc test; ** *p* < 0.01; *** *p* < 0.0001; ns, non-significant.
See also Figures S5–S7.</footnote>
A

<figure><img src="image_44.png" /></figure>

B

<figure><img src="image_45.png" /></figure>

C

AP sites in ssDNA

<figure><img src="image_46.png" /></figure>

D

AP sites in dsDNA

<figure><img src="image_47.png" /></figure>

tumors, especially following DNA damage.⁴⁷⁻⁴⁹ Accordingly, APOBEC3C and APOBEC3D have been recently shown to promote RAD51 loading onto replicating DNA in the absence of DSBs.⁴⁸

5hmdU is likely to be short lived due to its rapid formation and removal from ssDNA templates during DNA replication. This might explain the low levels of 5hmdU measured in total genomic DNA.⁷⁷

5hmdU formation might be stimulated by TET2-dependent accumulation of 5hmdC at under-replicated and damaged DNA⁷⁸ occurring in HR-defective backgrounds.⁷⁹,⁸⁰

Alternatively, 5hmdU could indirectly arise from APOBEC3-dependent deamination of 5mdC to yield 5mdU (dT), which can be oxidated to 5hmdU by TET2.⁷⁷ These events might take place in regions of high TET2 activity⁸¹ coupled to APOBEC3s enzymes. The complete characterization of the enzymatic events leading to AP sites from 5mdC awaits further clarification of these pathways.

Intriguingly, concurrent evolution of HR genes and appearance of DNA methylation in higher eukaryotes has been reported.⁸²

Overall, we established a direct connection between RAD51 and the protection of unrepaired AP sites from unregulated cleavage leading to fork breakage. In line with our findings, high levels of RAD51, commonly found in cancer cells⁸³ confer therapy resistance without completely restoring recombination in HR-defective cells.⁸⁴ Molecules targeting RAD51's ability to

<caption>**Figure 7. Model for BRCA2- and RAD51-mediated protection against AP site-induced fork breakage**</caption>

(A) Unrepaired AP sites stall nascent DNA synthesis. Restart by PRIMPOL leads to ssDNA gaps. RAD51 binds to unrepaired AP sites. Stabilized by BRCA2, RAD51 NPFs suppress AP sites cleavage by MRE11 and restrict further SMUG1 driven AP-site accumulation. Gap sealing is facilitated by POLθ and, possibly, TLS polymerases bypassing AP sites.

(B) Without BRCA2/RAD51, unrestricted SMUG1 access to ssDNA promotes AP sites and abasic gap accumulation. MRE11 extends ssDNA gaps and cleaves unprotected AP sites within ssDNA, especially in the absence of POLθ, causing replication fork breakage.

(C) APOBEC3B-dependent deamination of 5hmdC or 5mdC leads to transient 5hmdU formation, directly or indirectly. SMUG1 mediated removal of 5hmU promotes AP sites accumulation in ssDNA.

(D) TET2-dependent hydroxylation of 5mdC followed by TDG-mediated removal of the 5hmdC derivatives promotes the formation of AP sites in dsDNA repaired by BER.

protect AP sites might be designed to overcome resistance to therapy in RAD51 overexpressing cancer cells.

**Limitations of the study**

We could not find mutations in RAD51 that selectively eliminate its recognition of AP sites without also affecting RAD51's DNA-binding ability in the NPF as the DNA binding and interaction with AP sites depend on the same L1-L2 domains and residues in RAD51. Additionally, a possible role of RAD51 in repairing AP sites has not been defined yet.

**STAR★METHODS**

Detailed methods are provided in the online version of this paper and include the following:

* **KEY RESOURCES TABLE**

* **RESOURCE AVAILABILITY**

* Lead contact

* Materials availability

* Data and code availability

* **EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS**

* Xenopus laevis eggs

* Cell lines and culture

* Generation of DLD1 APEX1 knockout cell line

* Generation of DLD1 DNMT1<sup>NA</sup> and DNMT1<sup>NA</sup> DNMT3B<sup>-/-</sup> cell lines

* **METHOD DETAILS**

* Expression and purification of recombinant proteins

* Cloning

* DNA oligonucleotides for EMSA, SPR, and Cryo-EM

* Electrophoretic mobility shift assays (EMSA)

* Surface plasmon resonance (SPR)

* Vitrification of RAD51 filament grids

* CryoEM data collection and processing
* Real-space refinement

* Egg extract and chromatin binding

* *In vitro* enzymatic assays with DNA oligos

* DNA electron microscopy

* Chemical reagents

* Antibodies

* RNA interference

* Cell viability assays

* AP site quantification

* Western blotting

* Cell immunofluorescence

* DNA fiber assay with S1 nuclease

* DNA fiber assay with APE1 nuclease

* **QUANTIFICATION AND STATISTICAL ANALYSIS**
  * Statistical analysis

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.molcel.2024.07.004.

## ACKNOWLEDGMENTS

We thank members of the Costanzo lab for critical discussions, Federica Pezzi for technical support with Xenopus, Michele Giannattasio of the IFOM DNA EM facility for assistance with the EM, Silvia Monzani and Dr. Luigi Scietti of the Biochemistry and Structural Biology unit (BSU) at the IRCCS European Institute of Oncology (IEO, Milan, Italy) for the production and purification of the recombinant MR complex, and Melissa Milazzo for technical help. We thank Dimitri Chirgadze at the cryo-EM facility of the Department of Biochemistry of the University of Cambridge for support and advice with data collection and processing. The research leading to these results received funding from AIRC under IG 2023–ID. 28725 project, the Armenise-Harvard Foundation career development award, and an ERC grant (614541) awarded to V.C. M.A.R.-O. is supported by the 25318 AIRC fellowship for Italy. Y.W.H. is supported by the 26596 AIRC fellowship for Italy. Work in L.P. laboratory is funded by a Wellcome Trust investigator award (221892/Z/20/Z). R.A. is supported by a Cambridge BBSRC DTP studentship (grant number: 2271086).

## AUTHOR CONTRIBUTIONS

Y.W.H., M.A.R.-O., R.A., A.D.A., L.J., V.S., S.W., and A.A. performed all the experiments and analyzed the data. V.B. and D.F. produced the DNMTs cell lines and edited the manuscript. L.P. and V.C. analyzed the data, supervised the experiments, and wrote the manuscript. L.P. and V.C. are both corresponding authors as responsible for relevant complementary aspects of the work represented by structural and functional analysis, respectively. All authors agree that the contributions of M.A.R.-O., Y.W.H., and R.A are equal and that their positions on the author list can be swapped around on CVs, websites, and elsewhere if desired.

## DECLARATION OF INTERESTS

The authors confirm that there are no relevant financial or nonfinancial competing interests to report.

Received: March 20, 2024

Revised: June 3, 2024

Accepted: July 8, 2024

Published: August 22, 2024

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Mouse Anti-phospho-Histone H2A.X (Ser139) (IF: 1:800)</td><td>Merck/Sigma</td><td>Cat#05-636; RRID:AB_309864</td></tr><tr><td>Rabbit CCDC111/PRIMPOL Polyclonal antibody (WB: 1:2500)</td><td>Proteintech</td><td>Cat#29824-1-AP; RRID:AB_2918349</td></tr><tr><td>Rabbit Recombinant Anti-SMUG1 antibody [EPR15624] (WB: 1:1000)</td><td>Abcam</td><td>Cat#ab192240</td></tr><tr><td>Mouse mAb Anti-BRCA2 (Ab-1) (2B) (WB: 1:1000)</td><td>Merck/Sigma</td><td>Cat#OP95; RRID:AB_2067762</td></tr><tr><td>Rabbit anti-RAD51 (D4B10) WB: 1:1000)</td><td>Cell signaling</td><td>Cat#8875; RRID:AB_2721109</td></tr><tr><td>Mouse Monoclonal Anti-Vinculin WB: 1:20000)</td><td>Merck/Sigma</td><td>Cat#V9131-100UL; RRID:AB_477629</td></tr><tr><td>Purified Mouse Anti-MEK2 (WB: 1:1000)</td><td>BD biosciences</td><td>Cat#610235; RRID:AB_397630</td></tr><tr><td>Polyclonal Rabbit anti-Human APEX1 / APE1 Antibody (WB: 1:1000)</td><td>LsBio</td><td>Cat#Ls-C116767; RRID:AB_10916102</td></tr><tr><td>Rabbit Anti-TDG antibody [EPR8774] (WB: 1:1000)</td><td>Abcam</td><td>Cat#ab154192; RRID:AB_3096877</td></tr><tr><td>Anti-ATR</td><td>Aze et al.<sup>85</sup></td><td>N/A</td></tr><tr><td>Anti-MCM7 (Mouse monoclonal)</td><td>Santa Cruz</td><td>Cat#sc-9966</td></tr><tr><td>Anti-ORC1 (Mouse polyclonal)</td><td>Aze et al.<sup>85</sup></td><td>N/A</td></tr><tr><td>Anti-RPA70 (Rabbit polyclonal)</td><td>Aze et al.<sup>85</sup></td><td>N/A</td></tr><tr><td>Anti-CDC45 (Rabbit polyclonal)</td><td>Aze et al.<sup>85</sup></td><td>N/A</td></tr><tr><td>Anti-RAD51 (Mouse monoclonal) [14B4]</td><td>Abcam</td><td>Cat#ab123</td></tr><tr><td>Anti-Xenopus POL α p180 (Mouse monoclonal) peptide antigen used: VKRLPAVTKPGH</td><td>Kolinjivadi et al.<sup>32</sup></td><td>Abmart: clone 13026-1-3/C199</td></tr><tr><td>Anti-Xenopus POL δ 125 kDa (Mouse monoclonal) peptide antigen used: SSQTKKLRGDWDDD</td><td>Kolinjivadi et al.<sup>32</sup></td><td>Abmart: clone 19570-1-1/C316</td></tr><tr><td>Mouse anti-HMCES (B-2) (WB: 1:1000)</td><td>Santa Cruz</td><td>Cat#sc-514238; RRID:AB_2813859</td></tr><tr><td>DNMT1 (D63A6) Rabbit mAb (WB: 1:1000)</td><td>Cell signaling</td><td>Cat#5032; RRID:AB_10548197</td></tr><tr><td>Chicken anti-Rat IgG (H+L) Cross-Adsorbed, Alexa Fluor 488 (IF: 1:200)</td><td>ThermoFisher Scientific</td><td>Cat#A-21470; RRID:AB_2535873</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Alexa Fluor 568 (IF: 1:200)</td><td>ThermoFisher Scientific</td><td>Cat#A11011; RRID:AB_143157</td></tr><tr><td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed, Secondary Antibody, Alexa Fluor 568 (IF: 1:200)</td><td>ThermoFisher Scientific</td><td>Cat#A10037; RRID:AB_11180865</td></tr><tr><td>Rat monoclonal [BU1/75 (ICR1)] to BrdU (IF: 1:50)</td><td>Abcam</td><td>Cat#ab6326; RRID:AB_305426</td></tr><tr><td>Purified Mouse Anti-BrdU (B44) (IF: 1:50)</td><td>BD biosciences</td><td>Cat#347580; RRID:AB_10015219</td></tr><tr><td colspan="3"><strong>Biological samples</strong></td></tr><tr><td>Xenopus egg extract</td><td>This manuscript</td><td>N/A</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>APE1</td><td>New England Biolabs</td><td>Cat#M0282S</td></tr><tr><td>APE1 inhibitor III</td><td>Sigma-Aldrich</td><td>Cat#262017</td></tr><tr><td>Auxin, (Indole-3-acetic acid sodium salt, IAA)</td><td>Sigma-Aldrich</td><td>Cat#I3750</td></tr><tr><td>Mirin</td><td>Sigma-Aldrich</td><td>Cat#M9948</td></tr><tr><td>O-2-Propynylhydroxylamine hydrochloride, (AA3 probe)</td><td>Sigma-Aldrich</td><td>Cat#SYX00221-1G</td></tr><tr><td>PFM01</td><td>Sigma-Aldrich</td><td>Cat#SML1735</td></tr><tr><td>POLθi</td><td>Schrempf et al.<sup>21</sup></td><td>N/A</td></tr><tr><td>S1 nuclease</td><td>Sigma-Aldrich</td><td>Cat#N5661</td></tr><tr><td>S1 nuclease</td><td>Sigma-Aldrich</td><td>Cat#N5661</td></tr><tr><td>xSMUG1 protein</td><td>This manuscript</td><td>N/A</td></tr></tbody></table>

(Continued on next page)
**Continued**

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>xAPE1 protein</td><td>This manuscript</td><td>N/A</td></tr><tr><td>GST-BRC4</td><td>Hashimoto et al.<sup>14</sup></td><td>N/A</td></tr><tr><td>Geminin</td><td>Aze et al.<sup>85</sup></td><td>N/A</td></tr><tr><td>POLθ protein full length</td><td>Mann et al.<sup>15</sup></td><td>N/A</td></tr></tbody></table>

**Critical commercial assays**

<table><tbody><tr><td>CellTiter-Glo® Luminescent Cell Viability Assay</td><td>Promega</td><td>Cat#G7571</td></tr><tr><td>Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 488 dye, Alexa Fluor™ 647 dye</td><td>ThermoFisher</td><td>Cat#C10337; CHEBI:52661 Cat#C10340;</td></tr><tr><td>CRISPR Gene Knockout Kit v2 targeting APEX1</td><td>Sinthego</td><td>N/A</td></tr><tr><td>DNeasy Blood & Tissue Kit</td><td>Qiagen</td><td>Cat#69504;</td></tr><tr><td>Lipofectamine CRISPRMAX Transfection Reagent</td><td>ThermoFisher</td><td>Cat#CMAX00003;</td></tr><tr><td>Nucleostain- DNA Damage Quantification Kit -AP Site Counting</td><td>Dojindo</td><td>Cat#DK02-12;</td></tr><tr><td>Qubit™ dsDNA HS Assay Kits</td><td>ThermoFisher Scientific</td><td>Cat#Q32854;</td></tr><tr><td>RNAiMax</td><td>ThermoFisher</td><td>Cat#13778150;</td></tr></tbody></table>

**Deposited data**

<table><tbody><tr><td>Atomic Coordinates and CryoEM maps</td><td>Protein Data Bank (PDB) and<br/>Electron Microscopy<br/>Data Bank (EMDB)</td><td>PDB: 8RCD, 8RCF;EMDB: EMD-19050,<br/>EMD-19051</td></tr><tr><td>Images and graphs</td><td>Mendeley</td><td>Mendeley Data: https://doi.org/10.17632/2gjvrzddv7.1</td></tr></tbody></table>

**Experimental models: Cell lines**

<table><tbody><tr><td>Human DLD-1 wild-type</td><td>Horizon</td><td>Cat#HD PAR-008; RRID:CVCL_0248</td></tr><tr><td>Human DLD-1 BRCA2<sup>-/-</sup></td><td>Horizon</td><td>Cat#HD 105-007; RRID:CVCL_HD57</td></tr><tr><td>Human DLD-1 APEX1<sup>-/-</sup></td><td>This manuscript</td><td>N/A</td></tr><tr><td>Human DLD-1 DNMT1<sup>NA</sup></td><td>Scelfo et al.<sup>45</sup></td><td>N/A</td></tr><tr><td>Human DLD-1 DNMT3B<sup>-/-</sup> DNMT1<sup>NA</sup></td><td>Scelfo et al.<sup>45</sup></td><td>N/A</td></tr><tr><td>Human PEO1 BRCA2<sup>Y1655X</sup></td><td>Panzarino et al.<sup>41</sup></td><td>N/A</td></tr><tr><td>Human C4-2 PEO1 BRCA2<sup>WT</sup></td><td>Panzarino et al.<sup>41</sup></td><td>N/A</td></tr></tbody></table>

**Experimental models: Organisms/strains**

<table><tbody><tr><td>Xenopus laevis females</td><td>Nasco</td><td>LM00535MX</td></tr><tr><td>Xenopus laevis males</td><td>Nasco</td><td>LM00715MX</td></tr></tbody></table>

**Oligonucleotides**

<table><tbody><tr><td>siAPOBEC3B</td><td>Horizon</td><td>Cat#L-017432-00-0005</td></tr><tr><td>siBRCA2</td><td>Horizon</td><td>Cat#L-003462-00-0010</td></tr><tr><td>siHMCES</td><td>Sigma</td><td>Cat#EHU036741-5UG</td></tr><tr><td>siMRE11A</td><td>Horizon</td><td>Cat#L-009271-00-0010</td></tr><tr><td>siPRIMPOL</td><td>Horizon</td><td>Cat#L-016804-02-0005</td></tr><tr><td>siSMUG1</td><td>Horizon</td><td>Cat#L-012838-01-0010</td></tr><tr><td>siTDG</td><td>Horizon</td><td>Cat#L-003780-01-0010</td></tr><tr><td>siTET2</td><td>Horizon</td><td>Cat#L-013776-03-0010</td></tr><tr><td>Universal control siRNA</td><td>Sigma-Aldrich</td><td>Cat#SIC001</td></tr><tr><td>siRAD51</td><td>Horizon</td><td>Cat#L-003530-00-0010</td></tr></tbody></table>

**Recombinant DNA**

<table><tbody><tr><td>6His-pET-SMUG1</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pGEX-6P-1-APE1</td><td>This manuscript</td><td>N/A</td></tr><tr><td>Mre11 BACMID vector pTP2620</td><td>Bhaskara et al.<sup>33</sup></td><td>Addgene Cat#113311; RRID:Addgene_113311</td></tr><tr><td>Rad50 BACMID vector pTP813</td><td>Bhaskara et al.<sup>33</sup></td><td>Addgene Cat#113308; RRID:Addgene_113308</td></tr><tr><td>RAD51</td><td>Appleby et al.<sup>23</sup></td><td>N/A</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Software and algorithms</td></tr><tr><td>GraphPad Prism (version 9.3.0)</td><td>GraphPad</td><td>https://www.graphpad.com; RRID:SCR_002798</td></tr><tr><td>ImageJ version 2.3.0/1.53f</td><td>Schindelin et al.<sup>86</sup></td><td>https://imagej.net; RRID:SCR_003070</td></tr><tr><td>CellProfiler 3.1.9</td><td>McQuin et al.<sup>87</sup></td><td>https://cellprofiler.org; RRID:SCR_007358</td></tr><tr><td>Gatan Micrograph software</td><td>Gatan</td><td>N/A</td></tr></tbody></table>

Other

<table><tr><td>Bioruptor</td><td>Diagenode</td><td>N/A</td></tr><tr><td>Electron microscopy grids</td><td>Ted Pella</td><td>https://www.tedpella.com/grids_html/</td></tr><tr><td>ChemiDoc MP</td><td>BioRad</td><td>N/A</td></tr><tr><td>EnVision microplate reader</td><td>PelkinElmer</td><td>N/A</td></tr><tr><td>FEI Tecnai 20 EM microscope equipped with GATAN high-resolution camera</td><td>FEI+Gatan</td><td>N/A</td></tr><tr><td>MED20 evaporator</td><td>Leica</td><td>N/A</td></tr><tr><td>Leica TCS SP8-STED confocal microscope</td><td>Leica</td><td>N/A</td></tr><tr><td>Olympus Upright BX61 fluorescence microscope</td><td>Olympus</td><td>N/A</td></tr><tr><td>Stratalinker equipped with 254 and 365 nm ultraviolet light bulbs</td><td>Stratagene</td><td>N/A</td></tr><tr><td>TLA100 rotor</td><td>Beckman</td><td>N/A</td></tr></table>

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed and will be fulfilled by the lead contact, Vincenzo Costanzo (vincenzo costanzo@ifom.eu).

Materials availability

Materials generated in this study will be made available upon request by the lead contact.

Data and code availability

* The atomic coordinates and cryo-EM maps included in this study have been deposited in the Protein Data Bank and Electron Microscopy Data Bank (PDB 8RCD and 8RCF; EMD EMD-19050 and EMD-19051). Original western blot and microscopy images have been deposited in Mendeley Data: https://doi.org/10.17632/2gjvrzddv7.1. All datasets are publicly accessible as the date of publication.

* This paper does not report any original code.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Xenopus laevis eggs

Eggs derived from *Xenopus laevis* frogs were used as an experimental model system. Collection of eggs from the female frogs was performed in a non-invasive way following chorionic gonadotropin (Sigma, CG10) injections. Once collected eggs were processed to obtain cytoplasm, which was incubated with sperm nuclei to start DNA replication reactions. Occasional surgical procedures were performed on the male frogs to harvest sperm nuclei. Experimental protocols were approved by the IFOM Animal Welfare committee and the Italian Ministry of Health. The number of animals used was kept to a minimum and was calculated taking into account the number eggs required to obtain the cytoplasmic extract needed for the experiments described. The animals were kept in highly regulated and monitored conditions with room and water temperature at 19 °C. Basic husbandry requirements were provided by the IFOM XI facility.

Cell lines and culture

DLD-1 wild-type (DLD1WT) and DLD-1 BRCA2-deficient cells (BRCA2-/- cells,) were acquired from Horizon discovery. PEO1 cells bearing BRCA2Y1655X mutation and the corresponding PEO1 reverted cells (C4-2) expressing BRCA2WT protein were a gift from S. Cantor and T. Taniguchi. DLD-1, PEO1 and C4-2 cells were cultured in RPMI 1640 (Lonza) supplemented with 2 mM glutamine,
10% Fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen-Strep). Induction of DNMT1 degradation was archived by media supplementation with 500 µM of auxin (indol-3-acetic acid, IAA, SIGMA) for the time indicated in every figure. Cells were maintained at 37 °C and 5% CO₂, and were culture as described

**Generation of DLD1 APEX1 knockout cell line**

DLD1 APEX1 knockout cell line (hereinafter referred as DLD1 APEX1⁻/⁻) was generated by CRISPR-Cas9 technology. DLD1 APEX1⁻/⁻ cells were obtained by transient transfection of DLD1WT cells with synthetic sgRNAs targeting APE1 and Cas9 protein (Synthego) with Lipofectamine CRISPRMAX Transfection Reagent (ThermoFisher) following manufacturer's instructions. Seventy-two hours after transfections cells were seeded for clonal selection, expanded and positive clones were confirmed by western blot analysis sanger sequencing analysis.

**Generation of DLD1 DNMT1<sup>NA</sup> and DNMT1<sup>NA</sup> DNMT3B<sup>-/-</sup> cell lines**

DLD1 DNMT1<sup>NA</sup> and DLD-1 DNMT1<sup>NA</sup> DNMT3B<sup>-/-</sup> cells were previously described.<sup>45</sup> Briefly, DLD1 DNMT1<sup>NA</sup> cells were generated by electroporation of DLD1 WT and DNMT3B<sup>-/-</sup> cells expressing osTIR1-Myc9 with both the pX330 plasmid carrying a sgRNA targeting the ATG of DNMT1 and the donor template containing the mNeonGreen-AID tag by using the Lonza Nucleofector with Nucleofector Solution V following manufacturer's instructions. Five days after transfection, mNeonGreen positive cells were FACS sorted and seeded for clone isolation. Homozygous clones were expanded and confirmed by sanger sequencing, fluorescence microscopy and western blot analysis.

For DNMT3B knockout (DNMT3B⁻/⁻) DLD1 cells expressing both osTIR1-Myc9 and Tet-inducible Cas9-AID were transduced with pSB700-H2B-ECFP-lentivirus expressing a sgRNA targeting the ATG of DNMT3B. ECFP-positive cells were FACS sorted and seeded for clonal selection and expansion. After 10 days of doxycycline induction, DNMT3B⁻/⁻ clones were screened by PCR genotyping and immunofluorescence, and confirmed by western blot.

**METHOD DETAILS**

**Expression and purification of recombinant proteins**

Human wild-type RAD51 protein was expressed and purified as previously described.<sup>23</sup> Briefly, human RAD51 was co-expressed with His-MBP-BRCA2-BRC4 in *E.coli*. Cell pellets were resuspended in resuspension buffer (500 mM NaCl, 50 mM HEPES pH 7.4). Clarified cell lysate was loaded on a HisTrap HP 5 ml column pre-equilibrated with His Buffer A (20 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol) before isocratic elution in 40% His Buffer B (20 mM HEPES pH7.5, 300 mM NaCl, 5% glycerol, 500 mM imidazole). Eluate was diluted in A125 Buffer (20 mM HEPES pH7.5, 125 mM NaCl, 5% glycerol, 2 mM DTT) before loading on a pre-equilibrated HiTrap Heparin 5ml column and eluted in a gradient elution of 12.5% to 100% buffer A1000 (20 mM HEPES pH 7.5, 1 mM NaCl, 5% glycerol, 2 mM DTT). Fractions containing RAD51 were concentrated before a final purification step by size exclusion using a Superdex200pg gel filtration column pre-equilibrated with RAD51 storage buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 5% Glycerol, 2 mM DTT). Purified RAD51 was stored in small aliquots at -80°C.

Recombinant hMR was expressed and purified by the Biochemistry and Structural Biology Unit (BSU) at the European Institute of Oncology. The protein complex was expressed by co-infection of High Five Cells (BTI-TN-5B1-4) (ThermoFischer Scientific), with the baculovirus vectors pTP2620 encoding for hRAD50-HA-6His and pTP813 encoding for hMRE11-FLAG. The pTP2620 and pTP813 plasmids were a gift from Tanya Paull (Addgene #113311 and #113308 respectively). For protein purification pellets of infected cells were resuspended in lysis buffer (50 mM sodium phosphate pH 7, 500mM NaCl, 10% glycerol, 1mM DTT) supplemented with protease inhibitors cocktail Set III (Calbiochem), lysed by sonication and cleared by centrifugation at 70000 g for 45 min. The supernatant was loaded on a HisTrap HP column (Cytiva) using an AKTA chromatography system. The column was washed with a buffer containing 25 mM imidazole and was then eluted with an imidazole gradient from 25 mM to 500 mM in 10 column volumes. The resulting protein was concentrated and loaded on a Superose 6 column (Cytiva) pre-equilibrated in SEC buffer (50 mM HEPES, 150mM NaCl, 5% glycerol). Fractions containing monodisperse protein were concentrated in 50 kDa cut-off Amicon ultra centrifugal filters (Millipore) and flash frozen in liquid nitrogen for further assays.

POLθ protein purification was previously shown.<sup>15</sup> Briefly, Maltose Binding Protein (MBP)-tagged POLθ was expressed in insect cells following infection with the encoding baculovirus. Cell pellets were resuspended in MBP-lysis buffer (50 mM HEPES pH 7.6, 300 mM NaCl, 1 mM DTT, 1 mM EDTA) and cleared by centrifugation. The cleared lysate was loaded onto MBP-TRAP column (GE Healthcare), washed with 20 column volumes of washing buffer (50 mM HEPES pH 7.6, 300 mM NaCl, 1 mM EDTA, 5% glycerol); proteins were eluted with MBP-elution buffer (20 mM maltose, 50 mM HEPES pH 7.6, 200 mM NaCl, 1 mM EDTA, 5% glycerol). GST-BRC4 was purified as previously described.<sup>14</sup> Briefly, cDNA fragment encoding human BRC4 (amino acid 1511-1579 of BRCA2) cloned into pDONR221 (Invitrogen) were kindly provided by Dr F. Esashi (Oxford University). The fragment was then cloned into DEST15, an expression vector for GST-tagged recombinant proteins, using Gateway system (Invitrogen). The BRC4-DEST15 plasmid was transformed to BL21-AI cells, and recombinant GST-BRC4 protein production was induced by 0.2% (w/v) L-arabinose and purified with Glutathione Sepharose 4B according to standard procedures (GE Healthcare). GEMININ production was also previously described.<sup>85</sup> Briefly, 6xHistidine-tagged GEMININ (a gift from M Micheal) was expressed in BL21(DES) cells from a pET28 plasmid and purified on a nickel-NTA column (QUIAGEN) according to manufacturer instructions. GST-APE1 was
expressed in BL21(DE3) cells (Thermo Fisher Scientific. The protein expression was induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and at 37 °C for 3 hours. The cells were then harvested by centrifugation and resuspended in lysis buffer (50 mM Hepes/NaOH pH7.5, 150mM NaCl, 1mM DTT) supplemented with protease inhibitors cocktail setIII (Calbiochem), lysed by sonication and cleared by centrifugation. The cleared lysate was incubated with Glutathione-Sepharose 4B resin (Cytiva), for 1 hour at 4 °C and then were loaded on a bench chromatography column. The resin was first washed with 30 vol of wash buffer containing 50 mM HEPES pH7.5, 500mM NaCl and 1 mM DTT, then with two vol. of PreScission cleavage buffer (50mM Tris-HCl pH7.6, 150mM NaCl, 1mM DTT, 1mM EDTA), finally the protein was excised from GST using PreScission Protease (GST-HRV 3C). The eluted protein was further purified by SEC on a Superdex 75 10/300 column (GE Healthcare) in PBS buffer.

6His-pET-SMUG1 was expressed in BL21(DE3) cells. Cell pellets from cells expressing 6His-pET-SMUG1 were resuspended in His-lysis buffer (50 mM Hepes pH7.6, 300 mM NaCl, 10% glycerol, 2-mercaptoethanol 0.25% Triton X100) supplemented with protease inhibitors cocktail setIII (Calbiochem) and 12.5 U/ml Benzonase (Sigma), lysed by sonication and cleared by centrifugation. The cleared lysate was incubated with Talon metal affinity resin (Clontech) for 2 hours at 4 °C. The resin was then washed with 30 vol. of lysis buffer containing 500 mM NaCl and 10 mM Imidazole. The bound proteins were eluted with 300 mM imidazole in lysis buffer. The eluate was further purified by SEC on Superdex-75 column (GE Healthcare) preequilibrated in SEC buffer. Relevant fractions were concentrated in 50 kDa cut-off Amicon ultra centrifugal filters (Millipore).

## Cloning

Human wild-type RAD51 protein was cloned as previously described.²³ The cDNA sequences encoding X/APE1 and SMUG1 were obtained by RT-PCR, from RNA derived from X/ eggs with TRIzol Reagent (Thermo Fisher). The full-length APE1 sequence was amplified by PCR using Phusion High-Fidelity DNA Polymerase (Thermo Fisher) and the primers xAPE1-Bam-For and xAPE1-Xho-Rev (see table). The PCR product was cloned into pGEX-6P-1 expression vector (Invitrogen). The sequence was verified by sequencing using the primers listed in the table below. The full-length SMUG1 sequence was also amplified by PCR using Phusion High-Fidelity DNA Polymerase (Thermo Fisher) and the primers xSMUG1-For and xSMUG1-Rev (see table). The PCR product was digested with restriction enzymes BamHI and Xhol (New England) and cloned into pET43-6H vector (Invitrogen) obtaining the 6His-pET-SMUG1. The sequence was verified through sequencing process using the primers listed in the table below.

The BAC MID vectors pTP2620 and pTP813 to express recombinant wild type MRE11-RAD50 in Sf9 Cells used in this work were provided by Tania Paull through Addgene (Addgene #113311 and #113308, respectively)

<table><tr><td>xAPE1-Bam-For</td><td>GGGTTTGGATCCATGCCCAAGAGAGGGAAGAAGGAAAG</td></tr><tr><td>xAPE1-Xho-Rev</td><td>GGGTTTCTCGAGTCATATCGCCATAAGGAGTGTTATTGGA</td></tr><tr><td>xSMUG1-Bam-For</td><td>CGCGGATCCATGGCTGCGGAAGCTTGCGTG</td></tr><tr><td>xSMUG1-Xho-Rev</td><td>CCGCTCGAGCTATCAGCCAGTTAGTAAAGATAAAAC</td></tr></table>

## DNA oligonucleotides for EMSA, SPR, and Cryo-EM

PAGE-purified DNA oligonucleotides were purchased from IDT and resuspended to 100 µM in TE buffer. dsDNA was annealed in TE buffer with equimolar concentration of each constituent strand of a duplex to a final concentration of 10 µM, by boiling at 95 °C for 5 minutes followed by slow cooling to room temperature. The names, tag status and sequences of the oligonucleotides are reported below, together with an indication of the experiments where they were used. The underlined X in the oligonucleotide sequence marks the presence of an AP site.

<table><thead><tr><th rowspan="2">Name</th><th rowspan="2">Tag</th><th rowspan="2">Sequence (5' to 3')</th><th colspan="3">Experiment</th></tr><tr><th>EMSA</th><th>SPR</th><th>Cryo-EM</th></tr></thead><tbody><tr><td>AP0</td><td>Biotin</td><td>GGTATGCAGTGGTAGACGTGAGC</td><td>x</td><td>x</td><td></td></tr><tr><td>AP1</td><td>Biotin</td><td>GGTATGCAGTGXTAGACGTGAGC</td><td></td><td>x</td><td></td></tr><tr><td>AP3</td><td>Biotin</td><td>GGTATGCAXTGXTAXACGTGAGC</td><td></td><td>x</td><td></td></tr><tr><td>AP5</td><td>Biotin</td><td>GGTATXCAXTGXTAXACXTGAGC</td><td>x</td><td>x</td><td>x</td></tr><tr><td>AP7</td><td>Biotin</td><td>GGXATXCAXTGXTAXACXTGXGC</td><td></td><td></td><td>x</td></tr><tr><td>APc</td><td>-</td><td>GCTCACGTCTACCACTGCATACC</td><td>x</td><td></td><td>x</td></tr></tbody></table>

## Electrophoretic mobility shift assays (EMSA)

EMSA reactions were prepared in the following buffer: 25mM HEPES pH 7.5, 150 mM NaCl, 2 mM DTT, 2 mM ATP, 5 mM CaCl₂. The oligonucleotides used in the assay were: AP0 (ssDNA), AP5 (abasic ssDNA), AP0+APc (dsDNA), AP5+APc (abasic dsDNA). The DNA
was added to a final concentration of 500 nM and incubated with increasing amounts of RAD51 corresponding to final protein con-
centrations of 1, 2.5, 5, 10, 20, 30 µM. Reactants were incubated at 25 °C for 15 minutes prior to loading. Immediately before loading,
samples were mixed with a 0.5:1 volume of 50% (w/v) sucrose solution. Samples were run on a pre-run 0.5% agarose gel in 0.5xTB
buffer (45 mM Tris, 45 mM boric acid) (2.5hr, 4 °C, 35V). After running, the gel was stained in a 2x SYBR Gold solution made up in
MilliQ water (10 min, 25 °C), destained in MilliQ water (10 min, 25 °C), and imaged using a Typhoon FLA9000 by excitation at 473 nm.

Surface plasmon resonance (SPR)

Biacore T100 system (Cytiva) was washed twice with 1X HBS-E buffer (Cytiva) prior to coupling of the biotinylated DNA substrates on a Series-S sensor chip SA (Cytiva). The SA chip contains four flow cells that were used to couple the ssDNA oligos (AP0, AP1, AP3, AP5). The DNA oligos were prepared at final concentration of 100 nM in 150 µL of 1XHBS-E buffer. The DNA coupling process consisted of three injections with regeneration buffer (50 mM NaOH and 1 M NaCl) to activate the surface of the chip and remove any free or excess unbound streptavidin, followed by a pulse-mode injection of the DNA substrates into each flow cell at a flow rate of 10 µL/min and 10-second injection length. RAD51 samples at increasing final concentrations (100, 250, 500, 750, 1000, 2000, 3000, 5000 nM) were prepared in binding buffer (25 mM Hepes pH7.5, 100 mM NaCl, 5 mM CaCl₂, 5 mM MgCl₂, 1 mM ATP, 2 mM DTT) and injected sequentially from low to high concentration of RAD51. Binding response units were recorded at 20 µL/min for 240 seconds, injections were then stopped, a binding buffer wash started at the same flow rate and response units of dissociation were recorded for 360 seconds. DNA substrates and surface was regenerated by injecting regeneration buffer (25 mM Hepes pH 7.5, 1000 mM NaCl, 5 mM CaCl₂, 5 mM MgCl₂, 1 mM ATP, 2 mM DTT) at 100 µL/min for 120 seconds. The first-order association rate constants (K<sub>on</sub>) were calculated from the slope of the linear fit of the 250 nM RAD51 binding curve in the 20 to 90 second interval after injection. Each binding experiment of the RAD51 concentration series was performed in triplicate.

Vitrification of RAD51 filament grids

RAD51 protein was thawed on ice and used to prepare a reaction mixture for grid freezing containing a final concentration of 5 µM RAD51 in reaction buffer: 150 mM NaCl, 25 mM HEPES pH 7.5, 2 mM DTT, 2 mM ATP, 5 mM CaCl₂, supplemented with 250 nM of either abasic ssDNA (AP7) or abasic dsDNA (AP5+APc). Reactions were incubated for 15 minutes at 25 °C prior to vitrification. UltrAuFoil R1.2/R1.3 300 mesh gold grids (Quantifoil) were glow-discharged twice for 1 minute using a PELCO easiGlow system (0.38 mBar, 30 mA, negative polarity). 3 µl of reaction mix was applied to each grid before plunge-freezing in liquid ethane using a Vitrobot Mark IV robot (FEI), set to 100% humidity, 4 °C, 2 second blot time and -3 blot force.

CryoEM data collection and processing

RAD51 grids were screened using a Talos Arctica 200 keV cryo transmission electron microscope (TEM) fitted with a Falcon 3 de-
tector, and data were collected on a Titan Krios G3 CryoTEM fitted with a K3 detector using the EPU package (FEI). Grid preparation,
screening and data collection were all performed at the CryoEM facility in the Department of Biochemistry, University of Cambridge.
Data were stored and processed on the Cambridge Service for Data-Driven Discovery (CSD3) high-performance computer cluster
using Relion 3.1.⁸⁸ Data collection parameters are reported in Table 1. Micrograph motion correction was performed using
MotionCor2⁸⁹ and CTF estimation was performed using CTFFIND4.⁹⁰ Autopicking was performed based on manually picked 2D
class averages with a picking threshold of 0.1 and the number of asymmetric units in the filament box size set to 6 (approximately
one full turn of a filament). Autopicked particles were extracted with a box size of 200 Å for both CryoEM datasets and then binned
2x. 2D classification was performed iteratively on extracted particles with the option to ignore CTF correction until the first peak
selected in addition to processing using 'fast subsets'. For the RAD51 filaments with single-stranded abasic DNA, 183,769 high-res-
olution particles were identified following 2D classification, which were used to generate an initial 3D model. 3D classification was
performed with the helical reconstruction option turned on and starting values for the helical twist and helical rise of 56° and 16Å
respectively. All 3D classes showed clear density for single-stranded DNA in the filament core with a noticeable gap in the density
corresponding to the abasic site which repeated with every triplet. All 3D classes were selected, re-extracted and binned 2x. The
183,769 extracted particles were used to refine class 1 from 3D classification which was then globally sharpened in Relion 3.1 to
a final resolution of 3.22 Å. For the RAD51 filaments with double-stranded abasic DNA, 169,548 high-resolution particles were iden-
tified following 2D classification. Generation of an initial 3D model and 3D classification was performed as described for the single-
stranded DNA filament structure. All 3D classes showed clear density for duplex DNA in the filament core with a noticeable gap in the
density corresponding to the abasic site which repeated with every triplet. Classes one and three were selected to continue further on
the basis that they exhibited the clearest base-gap in the filament core. Selected particles from 3D classification were re-extracted
and binned 2x. The 8853 extracted particles were used to refine class one from 3D classification which was then globally sharpened
in Relion 3.1 to a final resolution of 3.43 Å.

Real-space refinement

Atomic models of the RAD51 NPFs with abasic ss- and dsDNA were built into the maps based on published structures of the pre- and
post-synaptic RAD51 filaments (PDB IDs 8BQ2 and 8BR2, respectively). Filament models with 7 RAD51-ATP chains, 14 Ca²⁺ and one
abasic DNA chain corresponding to the AP7 oligo (23 nucleotides), containing 7 regularly-spaced abasic sites (see DNA oligonucle-
otides for cryo-EM table); in addition, the filament model with abasic dsDNA contained the complementary DNA chain to AP7 (see
APc in DNA oligonucleotides for cryo-EM table). The model of abasic ssDNA NPF was refined against the post-processed map from Relion, whereas the model of the abasic dsDNA NPF was refined against the 3D refined map that had been sharpened using *phenix.local_aniso_sharpen* in PHENIX.⁹¹ Restrains for the abasic nucleotide were generated using the Grade Web Server at: https://grade.globalphasing.org/cgi-bin/grade2_server.cgi. Real-space refinement was interspersed with manual modelling in Coot⁹² to improve the fit of the model to the map. Refinement statistics²⁴,²⁵ for the two filament models are reported in Table 1.

## Egg extract and chromatin binding

X1 interphase egg extracts and sperm nuclei were prepared as previously described.⁸⁵ Briefly, X1 eggs were collected in MMR buffer (5 mM HEPES-KOH pH 7.5, 100 mM NaCl, 0.5 mM KCl, 0.25 mM MgSO₄, 0.5 mM CaCl₂, 25 µM EDTA) from chorionic gonadotropin injected female frogs. The eggs were de-jellied in 10 mM Tris pH 8.0, 110 mM NaCl and 5 mM DTT and rinsed three times in MMR. De-jellied eggs were released in interphase in presence of 5 µM Calcium Ionophore (A23187, Sigma) for 5-6 min, washed three times with MMR and rinsed twice in ice-cold S-buffer (50 mM HEPES-KOH pH 7.5, 50 mM KCl, 2.5 mM MgCl₂, 250 mM sucrose, 2 mM β-mercaptoethanol). Activated eggs were then packed by centrifugation at 1.200 rpm for 1 min and the excess of buffer was discarded. Eggs were crushed at 13.000 rpm for 12 min at 4 °C. The crude extract was collected and centrifuged at 70.000 rpm for 12 min at 4 °C in a TLA100 rotor (Beckman). The interphase extract was obtained by collecting and mixing the cleared cytoplasmic fraction together with the nuclear membranes. For sperm nuclei preparation 4 testis were removed from 2 male frogs and placed in petri dishes containing 10 mL EB buffer (50 mM KCl, 50 mM HEPES-KOH pH 7.6, 5 mM MgCl₂, 2 mM DTT). Testis were finely chopped with razor blade. The material was then transferred to 15 mL Falcon tube and spun at 2,000 x g, in a swinging bucket rotor for 5 min at 4 °C. The pellet was resuspended in a total volume of 2 mL of room temperature SuNaSp buffer (0.25 M sucrose, 75 mM NaCl, 0.5 mM spermidine, 0.15 mM spermine). To remove membranes 100 µl of 2 mg/ml lysolecithin (Sigma) were added and incubated for 10 min at room temperature. Reaction was stopped by adding 3% BSA (Sigma). The pellet was resuspended again in 2 mL EB and spun at 2,000 x g for 5 min at 4 °C. The final pellet was resuspended in 400 µl of EB + 30% glycerol. Sperm nuclei were tested for absence of DNA breaks with TUNEL assay as previously described.⁸⁵ Briefly, 20 µl of different sperm nuclei preparations (4000 n/µl) were incubated at 37 °C for 4 h in 170 µl H₂O supplemented with 20 µl 10 x TdT buffer (NEB), 90 U Terminal transferase (NEB) and 1 µl α⁻³²P-dCTP. Aliquots of the reaction were then precipitated with 5% TCA, 2% pyrophosphate solution and spotted on Whatman GF-C glass fiber filter. After ethanol washes, filters were dried and the incorporated TCA precipitable radioactivity was counted in scintillation counter. Sperm nuclei preparations with the lowest counts were used for all the experiments.

For chromatin binding 40 µl egg extract containing sperm DNA were isolated from master reactions treated as shown in Figure legends at the indicated time points. For immunoblotting, samples were diluted with 10 volumes of EB (100 mM KCl, 2.5 mM MgCl₂, and 50 mM HEPES-KOH pH 7.5) containing 0.25% NP-40 and centrifuged through a 0.5 M sucrose layer at 10,000 x g at 4 °C for 5 min. Pellets were washed once with EB and suspended in Laemmli loading buffer. Proteins were then resolved on an SDS-PAGE and monitored by WB.

## In vitro enzymatic assays with DNA oligos

### Enzymatic activity of xAPE1

All endonuclease experiments were performed *in vitro* using recombinant components.

100 nM of 5' fluorescein-labeled single-strand oligo substrates 5'FAM-AP0 and 5'FAM-AP1 containing an AP site at the position marked by X (see oligo table below) were dissolved in reaction buffer containing 10 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM MgCl₂, 1 mM DTT, and 5% of glycerol. DNA substrates and recombinant X1 APE1 protein were incubated for 5 min at 23°C. For these titration experiments, the reaction mix contained 5'FAM-AP0 or 5'FAM-AP1 DNA oligos and either buffer alone or dilutions of APE1 ranging from 25 nM to 400 nM. The reactions were then stopped by adding an equal volume of formamide loading buffer (95% formamide, 5 mM EDTA) and boiling for 5 min at 95°C. Samples were then resolved by running on 15% precast TBE-urea polyacrylamide denaturing gels (Criterion, Bio-Rad) at 100 V for 40 minutes. Gels were analyzed using the image processing software ImageJ.

### Enzymatic activity of xSMUG1

250 nM of 5' fluorescein-labeled single-strand oligo substrates 5'FAM-APU and 5'FAM-AP-AP were dissolved in reaction buffer containing 10 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM MgCl₂, 1 mM DTT, and 5% of glycerol. DNA substrates and increasing concentrations of recombinant X1 SMUG1 protein (produced for this project) were pre-incubated for 20 min at 23°C before adding 200 nM of recombinant xAPE1. The reactions were incubated for 10 further minutes before being stopped by adding an equal volume of formamide loading buffer (95% formamide, 5 mM EDTA) and boiling for 5 min at 95°C. Samples were then resolved by running on 15% precast TBE-urea polyacrylamide denaturing gels (Criterion, Bio-Rad) at 100 V for 40 minutes. Gels were analyzed using the image processing software ImageJ. For these xSMUG1 titration experiments, the reaction mix contained DNA oligos (5'FAM-APU and 5'FAM-AP-AP) and either buffer alone or dilutions of xSMUG1 ranging from 25 nM to 200 nM. Gels were analyzed using the image processing software ImageJ.

### Human MR- and APE1-mediated cleavage activity at AP sites

To test the MR AP-cleavage activity we used a method previously established.³⁴ Briefly, to obtain the AP substrate we used a 3'-end fluorescein labelled 50mer oligonucleotide (5'BIOT-3'FAM_50mer) (SIGMA) containing an uracil that was transformed in a AP site by treatment with uracil-DNA glycosylase (UDG) (NEB, M0282). 100 nM oligonucleotide was treated with 0.1 U/µl of UDG in UDG buffer (20 mM Tris, 1 mM DTT, 1 mM EDTA) for 10 min at 37 °C. 10µl of cleavage reaction containing 50 nM AP-oligonucleotide, 50-400 nM
MR in MR-reaction buffer (50 mM MOPS, 50 mM NaCl, 10 mM MnCl₂, 1 mM DTT) or 25-200 nM of recombinant human APE1 (APE1, NEB M0282) in NEB buffer-4, was incubated for 40 min at 37 °C. The reaction was ended by the addition of 1 µl of 10x STOP mix (2.5% SDS, 100 mM EDTA, 1 mg/ml Proteinase K) and incubation for 10 min at 37 °C. 10 µl of Gel Loading Buffer II (Thermo Fisher) containing 95% Formamide were added to the samples before separating the products on 15% Polyacrylamide-8M Urea gels. The signals were visualized with the iBRIGTH-1500 imaging system (Invitrogen) using the 455-485nm white EpiLED-illumination and the 508-557 emission filter. As standard-marker we used a 25 nM mix of the three fluorescein labelled oligonucleotides: 5'BIOT-3'FAM_U-50mer, 5'FAM_29mer and 3'FAM_20mer.

## RAD51 protection against MR- or APE1-mediated cleavage of AP sites

100 µM 5'BIOT-3'FAM_U oligonucleotide was first treated with 0.1 U/µl UDG in UDG buffer for 10 min at 37 °C, then we added increasing amount of human RAD51(0.5 - 4 µM) in the presence of 1 mM AMP-PNP in a buffer containing (20 mM Tris, 50 mM NaCl, 2 mM MnCl₂, 1 mM DTT) and incubated for further 20 min at 37 °C. Finally, we added MR at final concentration of 300 nM in MR-reaction buffer or 300 nM of recombinant human APE1 (APE1, NEB M0282) in NEB buffer-4 and incubated for further 40 min at 37 °C. The reaction was ended by the addition of the STOP mix and the samples were processed as described above.

## RAD51 protection against xSMUG1-mediated formation of AP sites

100 µM 5'FAM-APU_26mer oligonucleotide was first incubated with human RAD51(4 µM) in the presence of 1 mM AMP-PNP in a buffer containing (20 mM Tris, 50 mM NaCl, 2 mM MnCl₂, 1 mM DTT) or with buffer alone for 30 min at 37 °C. The reactions was supplemented with 50 nM xSMUG1 or buffer and further incubated for 30 min at 37 °C. The reactions was heat inactivated for 5 min at 95 °C. Finally, 400 nM xAPE1 was added and incubated for 30 min at 37 °C. The reaction was ended by the addition of the STOP mix and the samples were processed as described above.

<table><thead><tr><td>Name</td><td>Sequence (5' to 3')</td></tr></thead><tbody><tr><td>5'BIOT-3'FAM_U-50mer</td><td><b>BIOT</b>-CAGAAAGGGAAAGTATACAACAAAAGCAUCCTCAAGTCTTGG AGAGAACA-FAM</td></tr><tr><td>5'FAM_29mer</td><td><b>FAM</b>-CAGAAAGGGAAAGTATACAACAAAAGCA</td></tr><tr><td>3'FAM_20mer</td><td>CTCAAGTCTTGGAGAGAACA-FAM</td></tr><tr><td>5'FAM-AP0_36mer</td><td><b>FAM</b>-CAGTGCCGAAGCTTGTCAAGGTCCTGGAACTGACGT</td></tr><tr><td>5'FAM-AP1_36mer</td><td><b>FAM</b>-CAGTGCCGAAGCTTGTCAAGGTCCTGGAACTGACGT</td></tr><tr><td>5'FAM-APU_26mer</td><td><b>FAM</b>-CGCGAAACGCCTAGUGATTGGTAGGG</td></tr><tr><td>5'FAM-AP-AP_26mer</td><td><b>FAM</b>-CGCGAAACGCCTAGXGATTGGTAGGG</td></tr></tbody></table>

## DNA electron microscopy

DNA for electron microscopy analysis was processed as previously described with some modifications.<sup>13,32,93</sup> Briefly, for Xl replication intermediates preparation sperm nuclei (4000 n/µl) were incubated at 23 °C in 200 µl egg extract for 60 min, diluted with 400 µl of EB buffer, layered onto 800 µl EB-EDTA (EB buffer + 1 mM EDTA) + 30% (w/v) sucrose and centrifuged at 3,000 x g for 10 min at 4 °C. Pellets were resuspended in 100 µl EB-EDTA and transferred to a 96-well plate. 4,5',8-Trimethylpsoralen (TMP) was added at 10 µg/ml to each well. For human cells replication intermediates preparation 15x10<sup>6</sup> cells were collected. After standard trypsinization, the cells were transferred to 15 ml Falcon tubes and spun down at 600 x g for 5 min at 4°C. The cell pellets were then washed once with 5 ml ice-cold PBS, resuspended in 10 ml ice-cold PBS and transferred to 10x5 mm Petri dishes, to which 10 mg/ml of TMP (Trimethylpsoralen, Sigma-Aldrich) were added and mixed. For EM on human genomic DNA 10-15 x 10<sup>6</sup> cells were collected for each sample. The cells were trypsinized, transferred to 15 ml Falcon tubes, and centrifuged at 600 x g for 5 minutes. The cell pellet was then washed once with 5 ml of ice-cold 1X PBS, resuspended in 10 ml of ice-cold 1X PBS, and transferred to a Petri dish. Xl or human samples were incubated on ice for 5 min in the dark and irradiated with 365 nm ultraviolet light for 7 min on a precooled metal block. The procedure from TMP addition to irradiation with ultraviolet light was repeated four more times. Samples were then supplemented with 0.1% (w/v) SDS to lysate nuclei and treated with 100 µg/ml RNase A for 1 h at 37 °C. For complete protein digestion, psoralen-crosslinked chromatin was incubated with proteinase K (1 mg/ml) for 2 h at 50 °C. Genomic DNA was extracted by adding one volume of 1:1 (v/v) phenol-chloroform mixture, precipitated with isopropanol, washed with 70% ethanol and digested with 150 U PvuII HF restriction enzyme for 4 hours at 37 °C. To visualize DNA the samples were spread onto EM grids and stained as previously described.<sup>13,32,93</sup> Briefly, 50 ng DNA were resuspended in 5 µl of formamide supplemented with 0.4 µl of BAC solution (alkyl dimethyl benzyl ammonium chloride 0.2% w/v in formamide) diluted 1:10 (v/v) in water. The total volume was gently pipetted onto the surface of water in a Petri dish to form a film. DNA was transferred to carbon-coated EM grids (Ted Pella) by briefly allowing them to contact the surface of the DNA film using tweezers. After staining in 1% uranyl acetate solution followed by a brief wash in 100% ethanol, grids were air-dried on filter paper and then subjected to DNA carbon-platinum rotary shadowing with a Leica MED20. Image acquisition was obtained with a FEI Tecnai 20 EM microscope equipped with a GATAN high-resolution camera at the IFOM electron microscopy facility. Blind analysis of EM images was performed by EM specialists. Conversion of DNA length from nm to nucleotides was done considering 1 nt=0.34 nm under EM.
## Chemical reagents

Aphidicolin was used at a concentration of 3 µM (M-APH) as indicated in figure legends. POLθi was previously described.<sup>15,21</sup> PMF01 was purchased from SIGMA (SML1735). POLθi and PFM01 were used at the concentrations indicated in the text and figure legend.

## Antibodies

Anti-XI proteins antibodies were previously described.<sup>15,32,85</sup> Rat monoclonal [BU1/75 (ICR1)] antibodies to BrdU (ab6326) were purchased from Abcam. Purified Mouse Antibodies against BrdU (B44) (347580) were from BD bioscience. Mouse Anti-phospho-Histone H2AX-Ser139 antibody was from Millipore (05-636). Anti-histidine was obtained from Sigma (SAB4301134).

The primary antibodies used against human proteins were as follows (maker and dilution are indicated in parenthesis: Anti-BRCA2 (Millipore, 1:1000), anti-DNMT1 (Cell Signaling, 1:1000), anti-PRIMPOL (Proteintech, 1:2500), anti-MRE11 (Cell signaling, 1:1000), anti-SMUG1 (Abcam, 1:1000), anti-RAD51 (Cell signaling, 1:1000), anti-APEX1 (LSBio, 1:500), anti-phospho-Histone H2A.X (Ser139, Millipore, 1:1000), anti-MEK2 (BD biosciences, 1:1000), and anti-Vinculin (Sigma, 1:20,000), anti-Tet2 (Invitrogen, 1:1000), anti-HMCES (Santa Cruz, 1:1000), anti-APOBEC3B (Abcam, 1:1000), anti-TDG (Abcam, 1:1000). After incubation with primary antibodies, appropriate horseradish peroxidase-conjugated secondary antibodies were applied for detection.

## RNA interference

siRNA transfections were performed using Lipofectamine RNAiMax (ThermoFisher, 13778150) following manufacturer's instructions. The final concentration of siRNA was 25 nmol/L. The siRNAs used were Horizon smart pools against MRE11A (L-009271-00-0010), PRIMPOL (L-016804-02-0005), SMUG1 (L-012838-01-0010), TET2 (L-013776-03-0010), BRCA2 (L-003462-00-0010) and RAD51 (L-003530-00-0010), siTDG (L-003780-01-001), siAPOBEC3B (L-017432-00-0005) and HMCES (EHU036741-5UG) and universal control siRNA (SIC001) from Sigma. Knock down siRNA efficiency was assayed 48 hours after transfection by Western Blot analysis.

## Cell viability assays

Twenty-four hours after transfection cells were seeded in a 96-well plates at density of 0.5-2x10<sup>3</sup> cells/well. POLθI, APEi III or AR03 was added 24 hours later at the indicated concentrations. Five days after, viability was assessed with the CellTiter-Glo Luminescent Cell Viability Assay (Promega) following manufacturer's instructions. Relative cell viability (%) was expressed as a percentage relative to the untreated (DMSO) cells. Each viability experiment was performed at least three times.

## AP site quantification

AP sites in genomic DNA were quantified with DNA Damage Quantification Kit (Dojindo Molecular Technologies) following manufacturer's instructions. In brief, cells were treated as indicated and DNA was extracted with DNeasy Blood & Tissue Kit (Qiagen) supplementing the lysis buffer with RNase A at 1 µg/mL. Two micrograms of DNA were labeled with 5 mM of ARP probe 1h at 37 °C, purified through gel filtration and quantified using Qubit dsDNA HS (ThermoFisher). ARP in the labeled DNA was measured using an ELISA-like assay in a microtiter plate. The ARP-labeled DNA (30 ng in 60 µL) and 90 µL of DNA binding solution (Dojindo Molecular Technologies) were added to each well, and the plate was covered and incubated in the dark at 37 °C overnight. This was followed by incubation in 100 µL of HRP-Streptavidin solution (Dojindo Molecular Technologies) at 37 °C for 1 hour, and then Substrate Solution (Dojindo Molecular Technologies) at room temperature for 1 hour. Following addition of 100 µL of HCL 2N optical density (OD) measurements at 450 nm were then obtained from the wells. All ARP assays were performed in triplicate and the means were calculated. The data, expressed as the number of AP sites per 10<sup>5</sup> nucleotides, were calculated based on the linear calibration curve generated for each experiment using ARP-DNA standard solutions.

An alternative method based on AA3 as also used. Cells were seeded in 8-well chamber slides at 3 x 10<sup>4</sup> cells per well. The following day, cells were treated with AA3 (8 µM, Sigma-Aldrich), with or without APEi III (Sigma-Aldrich) or DMSO for 24 hours. To prepare the cells for immunofluorescence staining, they were pre-extracted with CSK100 buffer (100 mM NaCl, 10 mM PIPES pH 6.8, 3 mM MgCl<sub>2</sub>, 300 mM sucrose, 0.5% Triton X-100) supplemented with 1 µg/mL of RNase A for 5 minutes on ice. Cell were then fixed and permeabilized with 1% methanol:acetone (1:1) for 5 minutes. AA3 incorporated on DNA was labeled with Alexa Fluor 647 azide using click chemistry (Click-it EdU Cell Proliferation Kit, ThermoFisher) following manufacturer's instructions. After a final wash with PBS, coverslips were incubated with 5 µg/mL DAPI in PBS for 10 minutes to stain the cell nuclei. The coverslips were then washed once and mounted in Vectashield plus mounting medium (Vector Labs). The slides were stored at 4 °C until image acquisition. Images were acquired with a Leica TCS SP8-STED confocal microscope with a 60X oil immersion objective 1.35 NA or 40X oil immersion objective. At least 10 images were captured per condition and more than 500 nuclei were analyzed with CellProfiler 4.1.3. For AP site detection in XI nuclei were isolated through centrifugation in sucrose cushions and treated as above. ARP probe (Sigma A9723) was used instead of AA3.

Detection was obtained with Neutravidin DyLight 650 (Tehrmofisher 84607)

## Western blotting

Cells were collected by trypsinization and lysed in RIPA lysis buffer (150 mM sodium chloride, 1% NP-40, 0.5% sodium dodecyoleate, 0.1% sodium dodecyl sulfate, and 50 mM Tris, pH 8.0) supplemented with proteinase and phosphatase inhibitors. Whole-cell extracts were sonicated, quantified and boiled for 5 minutes at 55 °C in Laemmli buffer. Protein lysates were separated on precast
Midi-PROTEAN TGX 4%–20% gels (Bio-Rad). Subsequently, the proteins were transferred onto nitrocellulose membranes using the Trans-Blot Turbo system (Bio-Rad) and the high molecular weight program. The membranes were blocked with 5% milk in TBS + 0.1% tween for 1 hour. Following blocking, membranes were incubated overnight at 4°C with primary antibodies diluted in blocking buffer. After incubation with primary antibodies, appropriate horseradish peroxidase-conjugated secondary antibodies were applied for detection.

## Cell immunofluorescence

Immunofluorescence analysis was performed as previously described¹⁵ with minor modifications. In brief, cells were mock treated or treated with siRNAs against MRE11, BRCA2 or RAD51 as indicated in figure captions. Cells were then seeded on a glass coverslip or 8-well chamber slides, and after 24 hours, they were exposed to 2 µM POLθi, 10 µM PFM01 or APEI for 24 hours as indicated in figures. Following the drug treatment, cells were exposed to 10 µM EdU for 30 minutes. Subsequently, the cells were washed once with PBS and pre-extracted with CSK100 buffer (100 mM NaCl, 10 mM PIPES pH 6.8, 3 mM MgCl₂, 300 mM sucrose, 0.5% Triton X-100) for 2 minutes on ice. After another wash with PBS, the cells were fixed with 4% paraformaldehyde in PBS for 12 minutes at room temperature. Next, the cells were permeabilized with permeabilization buffer (0.2% BSA, 0.5% Triton X-100 in PBS) for 10 minutes, followed by a wash. The EdU-positive cells were detected using the Click-it EdU Cell Proliferation Kit (ThermoFisher) according to the manufacturer's instructions. The cells were then blocked with blocking solution (10 mM Glycine, 2% BSA, 0.2% gelatin, 50 mM NH₄Cl, 0.2% Triton X-100) for 1 hour at room temperature. Coverslips were incubated with the primary antibody for 1 hour at 37°C, followed by three washes with PBS. Subsequently, the coverslips/slides were incubated with the secondary antibody. After three additional washes with PBS, the coverslips were incubated with 5 µg/mL DAPI in PBS for 10 minutes, washed once, and mounted in Vectashield plus (Vector Labs). The mounted samples were stored at 4°C until image acquisition. Images were acquired with a Leica TCS SP8-STED confocal microscope with a 60X oil immersion objective 1.35 NA or 40X oil immersion objective. At least 10 images were captured per condition and more than 500 nuclei were analyzed with CellProfiler 4.1.3.

## DNA fiber assay with S1 nuclease

Cells were seeded in 6-well dishes, and after 24 hours, the cells were pulse-labeled with 50 µM CldU (Sigma) for 20 minutes. The cells were then washed twice with warm PBS and labeled with 250 µM IdU (Sigma) for an additional 40 minutes. During the second pulse, a POLθ or APE inhibitor III (APE1i) were added. Alternatively, cells were labeled 48 hours post transfection with Control or specific siRNAs. After labeling, the cells were trypsinized, counted, and resuspended in CSK100 buffer (100 mM NaCl, 10 mM PIPES pH 6.8, 3 mM MgCl₂, 300 mM sucrose, 0.5% Triton X-100) at a final concentration of 1 x 10⁶ cells/mL. The cells were permeabilized for 5 minutes at room temperature and then centrifuged at 7000 rpm for 5 minutes. The cell pellets were resuspended in 1 mL of S1 buffer (30 mM Sodium acetate pH 4, 2 mM Zinc sulfate, 50 mM NaCl, 5% glycerol). The exposed nuclei were incubated with 10 U/mL of S1 nuclease (Sigma) in S1 buffer. The nuclei were then centrifuged for 5 minutes at 7000xrpm, and the nuclei pellets were carefully washed once with 1 mL of PBS containing 0.1% BSA. After another centrifugation for 5 minutes at 7000xrpm, the nuclei were resuspended in PBS to a final concentration of 1-2 x 10³ nuclei/µL. Two microliters of the cell suspension were lysed on a clean glass slide with 8 µL of MES lysis buffer (50 mM MES pH 5.6, 0.5% SDS, 50 mM EDTA) for 6 minutes. Subsequently, the slide was tilted at a 15-degree angle to allow the DNA to spread. The slides were air-dried for 30 minutes, fixed in freshly prepared acetic acid/methanol (1:3) for 10 minutes, and then air-dried and stored at 4 °C overnight. To prepare for immunodetection, the slides were rehydrated with 1x PBS for 5 minutes. The DNA was denatured with 2.5 M HCl for 60 minutes, followed by several washes with PBS. The slides were then blocked in blocking solution (5% BSA, 0.2% Triton X-100 in PBS) for 20 minutes. Next, the slides were incubated with a primary antibody mix consisting of anti-BrdU antibody (Abcam 6326, recognizing CldU) anti-BrdU antibody (BD Biosciences, recognizing IdU) both at a dilution of 1:50, in blocking solution for 90 minutes at 37°C in a humid chamber. After incubation, the slides were washed once with PBS 0.1% Tween and twice with PBS for 3 minutes each. Subsequently, the slides were incubated with a secondary antibody mix consisting of donkey anti-mouse Alexa 568 (1:150, ThermoFisher) and chicken anti-rat Alexa 488 (1:150, ThermoFisher) in blocking solution for 45 minutes at 37 °C in a humid chamber. The slides were then washed three times with PBS, air-dried, mounted in Vectashield plus (Vector Labs), and stored at 4 °C until image acquisition. Images were acquired using a Leica Thunder widefield microscope with a 63X oil immersion objective (1.35 NA). Approximately 10 to 15 images were captured per condition, and at least 200 fibers were measured using ImageJ software (version 2.3.0/1.53f). The experiments were repeated at least two times.

## DNA fiber assay with APE1 nuclease

APE1 nuclease assay was essentially performed as the S1 assay with some modification. Permeabilized cells were centrifuged at 7000 rpm for 5 minutes and resuspended in 1mL APE1 buffer. Exposed nuclei were then incubated with 3 U/mL of recombinant human APE1 nuclease (NEB M0282) in APE1 buffer (50 mM Tris-HCl pH 8, 5 mM MgCl₂, 50 mM KCl, 5% glycerol) for 30 minutes at 37°C. Nuclei were centrifuged 5 minutes at 7000xrpm, nuclei pellets were carefully washed once with 1 mL of PBS 0.1% BSA, centrifuged 5 minutes at 7000 rpm and resuspend in PBS to a final concentration of 1-2x10³ nuclei/µL. DNA was spread and stained as is the DNA fiber assay.
# QUANTIFICATION AND STATISTICAL ANALYSIS

## Statistical analysis

All experiments, if not indicated otherwise in the figure legend, were performed three times and representative experiments are depicted. No statistical methods or criteria were used to estimate sample size or to include/exclude samples. Statistical analysis was performed with GraphPad PRISM software (version 9.3.0) and Microsoft Excel. Statistical differences in the DNA fiber length analyses were determined using Kruskal-Wallis test followed by Dunn's post hoc test for multiple comparisons calculated on the median values of independent experiment data. Unpaired t-tests were used to assess the difference in means of two groups of data. One Way ANOVA followed Dunnet's post hoc analysis was used to compare each of several groups means against a single control group while One Way ANOVA followed by Tukey post hoc test was used to assess pairwise comparisons between group means. In all cases, ns indicates not significant, \*P\<0.05, \*\*P\<0.01, \*\*\*P\<0.001 and \*\*\*\*P\<0.0001. Image analysis was conducted using ImageJ and CellProfiler 4.1.3. Statistical details for each experiment including sample size, significance values and tests are indicated in figure legends and figures.