Genome-wide screen reveals dependence of break induced replication on several distinct checkpoints

Domain: biology
Published: 2025-12-15
Editorial outcome: major_revision

Abstract
Break-induced replication (BIR) is a primary homologous recombination pathway for repairing one-ended double-strand DNA breaks, including those arising from collapsed replication forks and eroded telomeres. BIR frequently leads to loss of heterozygosity, genetic mutations, and gross chromosomal rearrangements, all hallmarks of cancer. Here, we conducted a genome-wide screen that allowed us to identify and validate the involvement of 33 novel yeast genes in BIR. We report that, while DNA damage and spindle checkpoint machineries are both required to delay nuclear division and provide adequate time for BIR to complete, the spindle position checkpoint is required to coordinate between nuclear division and cytokinesis. Furthermore, we show that two nucleopore proteins play a sequential role during BIR: Nup84 acts before DNA synthesis, while Nup188 functions later to support repair completion. Given the conservation of BIR between yeast and humans and the role of BIR in cancer development, human homologs of the identified BIR proteins may represent promising targets for anti-cancer therapeutics.

Introduction
Break-induced replication (BIR) is a homologous recombination pathway that repairs one-ended DNA double-strand breaks (DSBs), including those formed by telomere erosion and replication fork collapse. During BIR repair, one broken chromosome end primes DNA synthesis using the homologous donor as a template, and this repair synthesis can proceed for hundreds of kilobases (reviewed in refs. 1–4). The BIR replication machinery manifests as a “migrating bubble”, first synthesizing the leading strand, which subsequently serves as the template for lagging strand synthesis; consequently, inheritance of the newly synthesized DNA is conservative 5,6. The unusual mode of DNA synthesis during BIR causes high levels of genetic instabilities such as a mutation rate that is up to 1000 times higher than that of S-phase replication as well as frequent genome rearrangements and copy number variations (CNVs) 5,7–16. BIR has also been proposed as a source of genome instability in humans 2. In particular, BIR can induce segmental duplications found in precancerous cells 17, and, in the presence of DNA-damaging agents, BIR promotes the formation of mutation clusters similar to those observed in cancer 8,18,19. BIR is also responsible for alternative lengthening of telomeres (ALT) (reviewed in refs. 2,20–22) and for mitotic DNA synthesis (MiDAS) 23,24, which respectively maintain telomere length in ~15% of cancers and complete replication of so-called DNA fragile sites that remain unreplicated until mitosis.

Our knowledge of BIR in human cells heavily relies on decades of studies characterizing the pathway in yeast Saccharomyces cerevisiae, the organism in which eukaryotic BIR was initially discovered 25–29. Numerous proteins that promote BIR have been identified in yeast through the candidate gene approach. For example, factors involved in BIR synthesis including Pol δ-RFC-PCNA, Pif1, DNA primase-DNA polymerase α, and RPA were all identified in yeast 30–33 and confirmed to be conserved in humans 34,35. Pol32, which is a subunit of Pol δ that is not essential for S-phase DNA replication, is required for BIR 30, as is Pif1 helicase 32, and these two proteins have become important biomarkers to detect BIR in various experimental systems.

The genetic requirements for the first steps of BIR are similar to other homologous repair pathways: BIR is initiated by 5′-to-3′ resection executed by the Mre11-Rad50-Xrs2 complex, then relies on Dna2-Sgs1 helicases and/or Exo1 exonuclease 36–38, followed by the participation of Rad52, Rad51, Rad55, Rad57, and Rad54 to complete the homology search and displacement loop (D-loop) formation 26,28,39. Additionally, Srs2 helicase/translocase plays an important role by counteracting the formation of toxic intermediates that arise from the invasion of long Rad51-bound ssDNA accumulated behind the D-loop into the BIR template 40. Recent studies also revealed that BIR requires several histone modification proteins, including Hst3 and Hst4 histone deacetylases and the histone methyltransferase Dot1 41; however, the full complement of epigenetic enzymes involved in BIR remains unknown. Also, because BIR takes a significant amount of time to complete and usually occurs during G2/M or mitosis, the DNA damage checkpoint (DDC) machinery is required to initiate and maintain cell cycle arrest for BIR synthesis to complete prior to cell division 9,28. Recently, it was discovered that nucleopore proteins Nup84 and Nup60 as well as motor protein Kinesin-14 and the Slx5-Slx8 SUMO-targeted ubiquitin ligase are required for BIR 42–44, suggesting that successful completion of BIR may require relocation of the broken chromosome to the nuclear periphery.

Although the candidate gene approach has generated a notable list of genes that participate in BIR, our knowledge of the factors that contribute to successful execution and to regulation of BIR remains incomplete. To address this gap, in this study, we conducted a whole-genome screen using our new yeast BIR system and the yeast knockout (YKO) collection, which includes mutant strains with knockouts (KOs) of all non-essential genes, to identify new candidate genes required for BIR. As a result of this study, we identified and validated 33 previously unrecognized BIR driver genes encoding proteins in a variety of functional groups—including checkpoint proteins, nuclear pore structural proteins, and proteins involved in post-translational modification of nucleosomes—and subjected several of them to deeper functional analysis. We discovered that, in addition to the DNA damage checkpoint, successful completion of BIR requires coordination between nuclear division and cytokinesis mediated by the spindle assembly checkpoint (SAC) and spindle position checkpoint (SPOC), respectively.

Results
Yeast BIR system
We constructed an experimental system in yeast that allows for both quantitative and semi-quantitative assessment of BIR efficiency (Fig. 1a). This system is based on the haploid yeast strain KT556 (Supplementary Data 1), which contains an ADE2 gene and a 5′ portion of LYS2 gene (P-lys) containing an HO endonuclease recognition motif on chromosome V (ChrV) between CAN1 and NPR2. Notably, there are no essential genes located centromere-distal from this HO motif position. We also inserted a 3′ fragment of LYS2 (ys2), which shares 2.3 kb of homology with P-lys, into chromosome III (ChrIII) at the HIS4 locus (Fig. 1a). Addition of galactose to the medium induces GAL::HO endonuclease, leading to a DSB in P-lys that invades ys2 and repair by BIR. Consequently, successful BIR repair can be identified by an Ade-Lys+ phenotype (Fig. 1a).

Following DSB induction, the survival rate in wild-type (WT) cells was 46.5% (Fig. 1b), and 90% of survivors were Ade-Lys+ (~42% BIR efficiency). The chromosome structures of BIR outcomes were confirmed by contour-clamped homogeneous electric field (CHEF) gel electrophoresis followed by Southern blot analysis (Supplementary Fig. 1). Deletion of RAD51, RAD52, POL32, or SRS2, known to be critical BIR genes, significantly decreased BIR efficiency (Fig. 1b and Supplementary Data 2.1). In addition, we determined that BIR efficiency could be efficiently determined by the number of Lys+ papillae formed when replica-plated from complete medium onto synthetic complete medium lacking lysine (SC-Lys) (Fig. 1c), providing a rapid, semi-quantitative test for BIR efficiency. Together, these results indicate that our new ectopic BIR system is suitable for quantitative and semi-quantitative analysis of BIR efficiency.

Whole genome screen for BIR genes
To conduct a genome-wide screen for yeast genes that drive and regulate BIR, we modified our ectopic BIR experimental system by inserting several genetic markers to allow selection for GAL::HO and BIR cassettes, as well as to enable the selection of ectopic outcomes (see “Methods” and Supplementary Fig. 2). The resulting strain was used to perform a genome-wide screen by individual crossing of this MATa strain with strains from the YKO collection containing ~5000 MATα strains with individual non-essential genes disrupted by a KANMX cassette, conferring resistance to G418 (Fig. 1d and Supplementary Fig. 2).

Next, BIR efficiencies in the resulting KO strains were assessed using the semi-quantitative approach (Fig. 1c), where strains were first grown on YEPD plates (see “Methods”) to allow a low level of DSB induction associated with “leaky” expression of the GAL::HO endonuclease, followed by replica plating onto SC-Lys medium (Fig. 1d; “Methods”). We identified 335 KO strains (Supplementary Data 3) with fewer Lys+ papillae compared to WT, 19 of which (e.g. RAD52, RAD51, POL32, SRS2, RAD54;) were already known to promote BIR. From the rest, 92 genes were selected for further characterization based on their known roles in processes related to DNA metabolism, including, for example spindle checkpoints, nucleopore structure, and post-translational modification of proteins associated with DNA (Supplementary Data 3). The other identified genes, including mitochondrial and metabolism/biosynthetic genes (Supplementary Data 3), were expected to affect BIR indirectly and, therefore, were not prioritized in this study.

We re-introduced deletions of 64 of the 92 prioritized genes into our ectopic BIR system (AM3422), and we observed that 30 of these genes were indeed required for BIR (Fig. 1e), as their deletions significantly decreased BIR efficiency (Supplementary Data 2 and 3). In addition, we were able to introduce 24 of these 30 deletions in another ectopic BIR system (YSJ131), and each of these single-gene mutants showed BIR defect (Supplementary Data 2.2); in this system, the HO-induced break occurs in chromosome V, and BIR repair is initiated by invasion into leu2 on chromosome III, followed by 41 kb of repair synthesis 45 (Supplementary Fig. 3a). Twenty-six of the new KO strains did not show any BIR defect (Supplementary Data 2.1), indicating that deletion of these genes likely affected cell growth on SC-Lys without affecting BIR specifically. Interestingly, deletions of the remaining six genes (UBA3, UBA4, MUB1, CCS1, YVH1, and PIB2) resulted in increased BIR efficiency, suggesting these genes represent BIR suppressors; however, these genes were probably selected by our screen due to conferring a slow-growth phenotype (Supplementary Data 3).

Spindle assembly checkpoint genes are required for BIR completion
The major function of the spindle assembly checkpoint (SAC) is to ensure accurate chromosome segregation by monitoring for proper attachment of spindle microtubules to kinetochores. In the presence of unattached or incorrect kinetochore–microtubule attachments, the SAC is activated to delay nuclear division 46,47. Our screen identified a BIR defect in strains with deletion of the SAC genes BUB1 and BUB3, suggesting a role for the SAC in BIR. In support of this hypothesis, we observed a defective BIR in bub1Δ, bub3Δ, and mad2Δ mutants in two ectopic BIR systems (AM3422 and YSJ131; Fig. 2a, b and Supplementary Fig. 3a, b; Supplementary Data 2), However, no defect was observed upon deletion of the SAC gene MAD3 (Fig. 2a and Supplementary Data 2).

To investigate the mechanism by which SAC mutants affect BIR, we deleted SAC genes in our allelic BIR system 48, which is disomic for Chr III (Fig. 2c). In this system, galactose-induced DSBs in a truncated copy of Chr III (recipient) are repaired mainly by BIR using a full-length Chr III (donor) as the homologous template. Successful BIR repair results in copying of ~100 kb of template DNA and yields two full-length copies of Chr III. Auxotrophic markers allow determination of BIR (Leu-Ade+), while gross chromosomal rearrangements (GCRs), which also present phenotypically as Leu-Ade+, are distinguished from BIR by CHEF gel analysis (Supplementary Figs. 4–6). Phenotypes of other repair events are also distinguishable: gene conversion (GC) outcomes are Leu+Ade+; failed repair, including chromosome loss of the truncated Chr III (CL), presents as Leu-Ade-red; and half-crossovers (HCs) resulting from fusions between the recipient and donor chromosomes are characterized by Leu-Ade-white (Fig. 2c). An important feature of this strain is that it is usually viable even when repair fails.

Consistent with our findings in the other systems, BIR was significantly reduced in bub1Δ, bub3Δ, and mad2Δ mutants (Fig. 2d and Supplementary Fig. 4 for CHEF analysis), but not in mad3Δ mutants (Supplementary Data 4). In addition, bub1Δmad2Δ double mutants showed a BIR defect similar to each single mutant (Fig. 2d and Supplementary Data 4; Supplementary Fig. 4a, h). Together, our data support that the SAC plays an important role during BIR.

To determine the specific BIR step that is defective in SAC mutants, we examined the kinetics of BIR using our droplet digital PCR (ddPCR)-based Assay for Monitoring BIR Elongation Rate (AMBER) 49. AMBER showed that, in WT cells 10 h after DSB induction, DNA copy number had increased ~1.5x at the P2 position (~6 kb from the X-region of MAT locus), ~1.4x at P4 (~34 kb away), and ~1.4x at P5 (~65 kb away) (Fig. 2c, e and Supplementary Data 5, 6), in agreement with previous observations 49. However, the increase in copy number at these positions was lower in bub1Δ and mad2Δ than in WT (Fig. 2f, g and see also Fig. 2h for the comparison at P2), indicative of a decrease in BIR synthesis in these mutants.

SAC proteins are required to prolong G2/M arrest during BIR
Because BIR is a slow repair process that can take more than 10 h, the efficiency of BIR heavily relies on DNA damage checkpoint (DDC) activity, which initiates G2/M cell cycle arrest to provide sufficient time for BIR to complete prior to nuclear division 9,28. Based on the hits from our screen, we hypothesized that BIR may also require the SAC to prolong the cell cycle arrest established by the DDC. To test this, we created rad9Δbub1Δ, rad9Δbub3Δ, and rad9Δmad2Δ strains defective for both DDC and SAC activities and compared the effects of these double deletions on DSB repair with those of single ones using the Chr III disomic system. Consistent with previous findings 9, rad9Δ cells showed significantly reduced BIR efficiency, and double mutants with a defective DDC and SAC exhibited an even greater decrease in BIR efficiency (Fig. 3a and Supplementary Fig. 4), supporting our hypothesis that both the DDC and the SAC contribute to successful completion of BIR.

If the SAC is required to extend cell cycle arrest initially established by the DDC, we expected that insufficient SAC activity would shorten the duration of G2/M arrest in cells undergoing BIR, causing SAC mutants to complete nuclear division more rapidly. To test this, we monitored nuclear division in disomic BIR derivative strains using DAPI staining and determined the proportion of cells in different cell cycle phases for 12 h after DSB induction (Fig. 3b). In WT, prior to DSB induction, ~70% (on average) of cells were in G1, while G2/M arrested cells were rare (~6%) (Fig. 3c, i and Supplementary Data 7). Upon DSB induction, the proportion of G2/M cells rapidly increased, reaching a maximum of ~89% by 5 h post-DSB. At the 8-h time point, ~15% of cells had resumed nuclear division, and the fraction of G2/M-arrested cells had decreased, reflecting completion of BIR repair (Fig. 3c, i). Like WT cells, the majority of bub1Δ and mad2Δ cells successfully arrested at G2/M, with the maximum proportion of arrested cells achieved by 5 or 6 h post-DSB (Fig. 3d, e, i and Supplementary Data 7); however, the fraction of G2/M-arrested cells remained lower compared to WT between the 5 h and 8 h time points (Fig. 3i). This suggests that the SAC may play a role—in parallel with the DDC—in the establishment of G2/M arrest, which is consistent with the decrease in BIR synthesis observed by AMBER starting around 6 h post-DSB in SAC mutants (Fig. 2h). Further, resumption of mitosis was earlier as it was observed more frequently in mutants compared to WT cells between the 6.5 h and 8 h time points (Fig. 3j). These findings indicate that BIR requires participation of both the DDC and SAC checkpoints. Specifically, we propose that G2/M checkpoint arrest established in cells undergoing BIR repair is extended by the SAC, and that this is important for BIR completion.

To explore whether the SAC may contribute to the establishment of G2/M arrest in the absence of a functional DDC, we microscopically analyzed rad9Δ cells as well as rad9Δbub1Δ and rad9Δmad2Δ double mutants. In rad9Δ cells, the fraction of G2/M cells reached ~28% by 4 h post-DSB and a maximum of ~41% by 7 h post-DSB (Fig. 3f, k), demonstrating that these cells could undergo a partial G2/M arrest, consistent with previous reports 9. In rad9Δbub1Δ and rad9Δmad2Δ cells, the proportion of cells in G2/M remained low throughout the time course and was significantly lower compared to rad9Δ cells (Fig. 3g, h, k and Supplementary Data 7), supporting that checkpoint-mediated cell cycle arrest was abolished in the double mutants and suggesting that the partial G2/M arrest in rad9Δ is driven by SAC machinery. Overall, we propose that, in addition to prolonging a DDC-induced G2/M cell cycle arrest, the SAC can mediate the establishment of cell cycle arrest in response to a DSB in the context of both a functional and a defective DDC.

The spindle position checkpoint is essential for BIR
Given that BIR proceeds during an extended G2/M arrest, wherein the undivided nuclei are positioned at the bud neck, we posited that BIR may also require the spindle position checkpoint (SPOC; reviewed in ref. 50) to block mitotic exit until the nucleus migrates into the bud after BIR is completed. To test the dependence of BIR on the SPOC, we deleted the main SPOC gene, BUB2 in two ectopic BIR systems, one of which required 65 kb (AM3422) and the other required 41 kb (YSJ131) of repair synthesis to complete BIR and observed that deleting BUB2 led to decreased BIR efficiency in both systems (Supplementary Fig. 3a–c). In the allelic (disomic) system, the kinetics of BIR synthesis measured by AMBER in a bub2Δ mutant was similar to that observed in the SAC mutants (Figs. 2h and 4a, b). However, strikingly, bub2Δ mutants exhibited significantly low viability after the DSB in this disomic system, in which a BIR defect normally does not lead to cell death (Fig. 4c and Supplementary Data 4): only 4% of bub2Δ cells survived after the DSB compared to > 95% viability in WT and SAC mutants. Microscopic examination demonstrated that, following plating on YEP-Gal medium, most bub2Δ cells divided no more than 2-3 times, and no cells formed micro-colonies after DSB induction (Supplementary Fig. 3d), confirming lethality following the DSB. A similar striking loss of viability after BIR induction was observed when BFA1, another SPOC gene encoding a partner of Bub2 51,52, was deleted (Fig. 4c). In addition, genetic analysis demonstrated that both SPOC mutants exhibited a decreased fraction of BIR outcomes among the rare survivors and a significant increase in GC outcomes (Fig. 4d and Supplementary Fig. 5), which concludes faster than BIR and usually does not trigger checkpoint arrest in the allelic system 28. However, in an ectopic system (YSJ133) 45, where the homology search takes longer and even GC can trigger checkpoint arrest BUB2 deletion led to a modest decrease in GC efficiency (Supplementary Fig. 3b), but the defect of BIR in this system (YSJ131) 45 was more severe (Supplementary Fig. 3b). The fraction of HC outcomes among survivors was very low in bub2Δ and bfa1Δ mutants (Fig. 4d), likely because of the extended time required for HC formation, which involves secondary breakage of the donor chromosome. Collectively, these results support that the viability of cells undergoing BIR depends on the SPOC.

To determine the mechanism underlying the dependence of cells on the SPOC during BIR repair, we monitored nuclear divisions in SPOC mutants. We observed that, similarly to SAC mutants, bub2- and bfa1-deficient cells started recovery from G2/M arrest earlier, at 5–6 h after DSB induction (Fig. 4e–g, i and Supplementary Fig. 3e), compared to WT. Notably, though, starting from 6 h, SPOC mutants accumulated cells with a unique morphology: they re-budded and formed “chained” cells (usually groups of 3 cells) containing only one undivided nucleus (Fig. 4e–g, j). We propose that the premature re-budding and cell death result from the inability of SPOC-deficient cells to delay their cytokinesis in response to the nuclear division arrest caused by the DDC and SAC. Indeed, deletion of MAD2 in bub2Δ mutants led to earlier escape from G2/M arrest after DSB induction (Fig. 4h, i). Consequently, compared to bub2Δ, the double mutants showed a significantly decreased occurrence of “chained” cells (Fig. 4j), increased cell viability (Fig. 4c), and a higher fraction of interrupted BIR outcomes, including HC and CL among survivors, making the double mutants phenotypically similar to mad2Δ (Figs. 2d and 4d). Altogether, these results support our hypothesis that coordination between nuclear division and cytokinesis is critical for the success of BIR and for viability of cells undergoing BIR. Consistent with this notion, three other genes involved in nuclear migration (JNM1, NUM1, and ARP1) 53,54 and known to have genetic interactions with SPOC genes 55 were identified in this BIR screen and validated as genes required for BIR in our ectopic system (AM3422; Supplementary Fig. 3f).

Differential roles of inner and outer ring nuclear pore proteins in BIR
It has been proposed that, similar to other DSB repair events that take a significant amount of time for completion, BIR requires chromosomes to re-locate to a nuclear pore to execute repair (reviewed in ref. 56). NUP84, encoding a component of the outer nuclear pore ring, was previously reported to be important for BIR 42,43, but the precise step of BIR where it functions has not been determined. In addition, our screen identified a dependency of BIR on NUP188, which encodes a protein of the inner ring of nuclear pores 56.

To increase our understanding of the role of nuclear pore proteins during BIR, we compared nup188Δ and nup84Δ mutants and found that both mutants exhibited a BIR defect in our ectopic system (Supplementary Fig. 6a). In the allelic system, loss of either gene also led to profound BIR defects; however, the specific phenotypes following DSB induction observed in these two mutants differed from each other. Specifically, in nup84Δ, the fraction of Leu-Ade+ outcomes was decreased and the frequency of defective DSB repair (i.e., CL) was increased, while cell viability remained high and similar to WT (Fig. 5a, b). In addition, CHEF analysis demonstrated that a very large fraction (32%) of Leu-Ade+ outcomes in nup84Δ cells represented GCRs (Supplementary Fig. 6b, c), supportive of a BIR defect in the absence of Nup84. Moreover, based on AMBER, even initiation of BIR synthesis was defective in nup84Δ, with virtually no DNA copy number increase at any of the probe positions (Fig. 5c, e; compared to WT in Fig. 5d and Supplementary Fig. 6h).

To test the hypothesis that broken DNA ends may physically relocate to nuclear pores, we performed chromatin immunoprecipitation (ChIP), which showed that Nup84 accumulates at the DSB position 4 h after the break, and diminishes by 10 h (Fig. 5g). Next, using an anti-Rad51 antibody, we observed a WT level of Rad51 accumulation at the DSB in the absence of Nup84, suggesting that DSB resection and Rad51 loading proceed normally in the absence of Nup84 (Fig. 5h). Further, we used donor-specific primers that recognized a sequence 100 bp telomere-proximal to MAT, and we found that Rad51 loading on the donor (indicative of strand invasion) was decreased ~2 fold but still occurred in the absence of Nup84 (Fig. 5h). From these findings, we propose that while Nup84 facilitates strand invasion, and possibly the homology search, its essential role is in the next step of recombination, which could include D-loop stabilization and/or initiation of DNA repair synthesis.

Our results suggested that Nup188 acts later during BIR than Nup84. Specifically, AMBER showed that nup188Δ had only a very slight defect in the initial steps of BIR synthesis but a very dramatic defect at the end of synthesis (Fig. 5f and Supplementary Fig. 6h). We also found that nup188Δ had drastically decreased viability compared to WT (12% vs. 96%, respectively) but did not have a significant increase of GCRs among Leu-Ade+ outcomes (Fig. 5a, b and Supplementary Fig. 6b, d). We found that nup188Δnup84Δ double mutants had higher viability than nup188Δ (78% vs 12%, respectively) (Fig. 5a) and an altered distribution of DSB repair outcomes (Fig. 5b and Supplementary Fig. 6b, g), supporting our hypothesis that Nup188 participates later than Nup84 in BIR. In addition, it has been shown that human homolog of yeast Nup188 (hNUP188) interacts with centrosomes, the functional counterpart of spindle pole body 57, suggesting that Nup188 could be involved in checkpoint regulation. In support of this, the fractions of DSB repair outcomes that take a significant amount of time to complete (including, HC, and CL) were decreased in nup188Δ (Fig. 5b), similar to SPOC mutants. Additional support came from the observation that deleting MAD2 or RAD9 in nup188Δ significantly increased viability after DSB induction, albeit BIR remained defective (based on the distribution of repair outcomes) (Fig. 5a, b and Supplementary Fig. 6b, e, f). Together, we characterized the important role played by Nup84 during BIR initiation and identified Nup188 as a protein that is important for the successful completion of BIR.

Epigenetic modifiers regulate BIR
The discovery that histone deacetylases Hst3 and Hst4 and histone methyltransferase Dot1 are important for BIR 41 revealed the importance of histone modifications in BIR, even though their exact role in regulating BIR remains unclear. Our screen identified genes encoding both known (Dot1) and previously unrecognized protein-modifying enzymes that are required for high BIR efficiency, including proteins involved in acetylation, deacetylation, methylation, ubiquitination, and urmylation (Fig. 6a and Supplementary Fig. 7a; Supplementary Data 2, 3). While the specific substrates affected by these proteins during BIR will be explored in future studies, our initial analysis suggests that multiple pathways and complex mechanisms are involved.

Based on characterization of hits from our screen, we determined that the initiation of BIR involves the histone methyltransferase Dot1 as well as Doa1, a protein involved in ubiquitination-mediated proteolysis. Deletion of the corresponding genes significantly impaired BIR in the ectopic (AM3422) (Fig. 6a and Supplementary Data 2) and allelic systems (Fig. 6b, c) and caused a synthesis defect during BIR initiation, as detected by AMBER (Fig. 6d, e, h). During later steps of BIR synthesis, we observed that two different Rpd3 deacetylase complexes—small-Rpd3S and large-Rpd3L (see in ref. 58)—have opposing roles in BIR. In particular, Rpd3L was determined to be important for BIR completion, while Rpd3S, which shares several common subunits with Rpd3L, was observed to inhibit BIR. These conclusions were supported by our finding that deleting RPD3 or UME1 (encoding common subunits of the Rpd3L and Rpd3S complexes) as well as CTI6 or SAP30 (specific to Rpd3L) reduced BIR efficiency (Fig. 6a), while deleting EAF3 (specific to Rpd3S) increased it (Fig. 6a). We further observed that the absence of Sdc1, which participates in histone 3 lysine 4 trimethylation (H3K4me3) 55 known to be involved in the recruitment of Rpd3L 59,60, decreased BIR efficiency (Supplementary Data 2). Finally, despite the significant defect in formation of BIR outcomes observed using genetic methods (Fig. 6c), BIR kinetics measured by AMBER in rpd3Δ and cti6Δ mutants was similar to WT (Fig. 6f–h). We therefore propose that Rpd3L is involved in the formation/stabilization of the final BIR product.

Further, discovering Eaf1—a component of the NuA4 histone acetyltransferase (HAT) complex 61—in our screen uncovered an important role for the NuA4-SWI/SNF axis in BIR. We specifically observed that deleting EAF1 caused a significant BIR defect in the ectopic system (AM3422; Fig. 6a). It is known that NuA4 recruits the SWI/SNF complex to DSBs 62, and, consistently, deletion of SNF6 and SNF12, which were identified in our screen, also caused a BIR defect (Fig. 6a). Interestingly, not every subunit of the NuA4 HAT complex played the same role in BIR. For instance, deleting EAF5 did not impact BIR (Supplementary Data 2), while deleting YAF9 increased BIR efficiency (Fig. 6a). Because Yaf9 is also a member of the SWR1 complex, we hypothesized that the yaf9Δ phenotype may be driven by its role in the SWR1 complex. Consistent with this idea, deleting HTZ1, which encodes histone variant H2A.Z deposited around DSBs by the SWR1 complex 63, mimicked the yaf9Δ phenotype, with htz1Δ cells showing increased BIR efficiency (Fig. 6a), tentatively suggesting that, at least within this experimental system, SWR1 may suppress BIR by promoting histone H2A.Z deposition after a DSB.

Other pathways that participate in BIR
Our screen also identified BIR candidate genes belonging to several other functional groups (Supplementary Fig. 7b and Supplementary Data 3). We observed that deletions of several genes involved in protein sorting and trafficking complexes impaired BIR. Also, deletions of the following genes involved in the cellular stress response significantly negatively affected BIR efficiency: SOK2, SLG1, SLT2, PHO85. Similarly, a significant reduction in BIR efficiency was observed following the deletion of MMS19, which encodes an iron-sulfur cluster assembly protein and was previously shown to affect fragility associated with inverted DNA repeats 64. Further functional characterization of these genes and their related protein complexes is needed to delineate their precise roles in BIR.

Methods
Strains
Three experimental systems were used in this study to investigate BIR (see Supplementary Data 1): two haploid BIR systems—one based on yeast strain AM3422 and the other on strain YSJ131 45—where BIR proceeds through an ectopic position, and a disomic Chr III system (AM1411) in which DSBs are repaired by allelic BIR. We also used a YSJ133 45 which is isogenic to YSJ131 strain to conduct GC assay (see Supplementary Fig. 3a for details). AM1411 is a derivative of our BIR experimental strain, AM1003, which contains a truncated copy of Chr III that can be cut by a galactose-induced HO endonuclease at MATa and a full copy of Chr III that cannot be cut, as its Yα at MAT locus was deleted by the insertion of lys2-A7. AM1411 has the following genotype: MATa-LEU2-tel/MAT::lys2:A ade1 met13 ura3 leu2-3,112/leu2 thr4 hmlΔ::ADE1/hmlΔ::ADE3 hmrΔ::HPH ade3::GAL-HO FS2Δ::NAT/FS2 lys2Δ.

AM3422 is a new haploid BIR system, a derivative of KT556, which has the following genotype: MATa bar1Δ his7-2 trp1Δ ura3Δ leu2-3,112 ade2Δ lys2Δ cup1Δ yhr054cΔ cup2Δ V29616::CUP1 V34205::ADE2-HOcs-lys2Δ3’. This strain contains the ADE2 gene and a 5′ portion of LYS2 on Chr V between CAN1 and NPR2 genes. Importantly, there are no essential genes located centromere distal to this locus. The 3′ part of LYS2 is replaced with a modified version of the 117-bp HO endonuclease recognition cut site (see Supplementary Data 5 for the full sequence). This sequence allowed an efficient cutting (Supplementary Fig. 8), thereby promoting efficient BIR (Fig. 1b), but also allowed an optimal number of Lys+ papillae in a semi-quantitative BIR test (Fig. 1c), with no prior accumulation of Lys+. The 3′ part of the LYS2 gene (3812 bp) marked by TRP1 gene was inserted into HIS4 (position 65,934 to 68,333 bp of Chr III according to SGD). The two fragments of LYS2 (located on Chr V and Chr III) share 2339 bp of homology. Finally, the strain contains GAL::HO inserted into the ADE3 gene, while the HO gene at its native locus is deleted.

To construct the AM3874 strain used for the genome-wide screen for BIR regulators, the following modifications were made to AM3422. A NATMX cassette was inserted into the HIS4 locus 160 bp centromere distal to the lys2 fragment. The LEU2 gene at its native position in Chr III was replaced by URA3, and LEU2 was inserted into the AVT2 gene next to CAN1 to mark the 5′ part of lys2 on Chr V. Also, his7-2 was replaced by the wild type HIS7. The HIS4 gene was inserted into the native LYS2 locus on Chr II. This allowed for selection against the LYS2 allele of the YKO strains by selecting for His+. In addition, HIS3 was deleted at its native location by the delitto perfetto approach 81, while MFA1 was replaced by MFA-HIS3 (similar to refs. 64,82). We introduced a fragment containing the rpl28Q38K mutation (similar to refs. 64,82) by transformation using selection for resistance to cycloheximide. Finally, URA3 inserted at LEU2 was fully deleted while the URA3 gene was inserted next to (858 bp from) the ade3::GAL::HO cassette on Chr VII.

All single-gene deletion mutants were constructed by transformation with a PCR-derived KANMX, NATMX, HPHMX, or BleoR module, each flanked by sequences homologous to the regions surrounding the open reading frame of each gene 83,84. All constructs were confirmed by PCR and by phenotype. The bub1Δ, bub3Δ, mad2Δ, bub2Δ, bfa1Δ, and nup188Δ mutants were also tested by droplet digital (dd) PCR for the correct number of each chromosome, to make sure that these mutants were not aneuploids. See Supplementary Data 1 for the genotypes of all strains used in this study.

The strains used for ChIP analysis were generated by inserting a 13xMyc epitope tag, marked by KANMX at the C-terminus of the NUP84 and NUP188 coding sequences in the allelic BIR strain (Supplementary Data 1). The derivative strains were analyzed by BIR assay to confirm that the insertion of Myc tag did not affect the functions of Nup84 and Nup188 proteins (see Supplementary Data 4).

Media and growth conditions
Rich media, Yeast extract-peptone-dextrose (YEPD), YEP-Raffinose (YEP-Raf), YEP-Galactose (YEP-Gal), and YEP-Lactate (YEP-Lac) media were prepared as described 85. Antibiotics, including hygromycin, nourseothricin, G418, have been added to YEPD as described 85. Synthetic complete media with bases and amino acids omitted (SC-Leu, SC-Ura, SC-Ade, etc) were prepared as described 85. Sporulation medium with potassium acetate (KAC) was prepared as: 2% potassium acetate, 2.5% Agar, pH = 7. All yeast cultures were grown at 30°C.

BIR efficiency assay in the yeast ectopic systems
Cells of yeast strains including AM3422, YSJ131 and their derivatives were cultured in 2 ml YEP-Lac liquid media with aeration for 18–24 h until reaching a concentration of ~2×107 cells/mL. Next, the cultures were diluted and plated on YEPD and YEP-Gal media, at ~200 cells/plate. The colonies were counted after 3–4 days of incubation and the viability following the DSB was calculated as a ratio of the colony forming units (cfu) on YEP-Gal to YEPD. The difference in viability after DSB in mutant strains and WT was compared by one-way ANOVA (Supplementary Data 2). To determine the fraction of BIR repair among DSB repair outcomes, the colonies obtained from AM3422 and its derivatives grown on YEP-Gal (and YEPD as a control) were replica plated to SC-Ade and SC-Lys plates. The fraction of BIR was calculated as the fraction of Ade+ Lys-.

The chromosome structure of BIR repair outcomes was confirmed by CHEF gel electrophoresis followed by Southern blot analysis using CHEF-DRII (Bio-Rad) apparatus. The preparation of DNA for CHEF in agarose plugs was performed as previously described in ref. 85. The running conditions for CHEF were optimized for the maximal separation of Chr V and Chr III, as follows: initial switch time of 35.43 s, final switch time of 52.33 s, running time of 39 h, and voltage of 6 Volts/cm. Following separating by CHEF, the DSB repair products were hybridized to P32-labeled probes specific to MGR1 (on the left arm of Chr III) and to GLN3 (on the right arm of Chr V) (see Supplementary Data 5 for the sequences of oligonucleotides used to create probes). Prior to BIR, the MGR1 (Chr III)-specific probe detected a 337 kb-long Chr III. After BIR, the MGR1 probe detected both the original Chr III and a Chr V/Chr III recombinant chromosome, which was 619 kb in size. The latter product was also hybridized to the GLN3 (Chr V) specific probe.

BIR assay in the yeast allelic (disomic chromosome III) system
Cells of yeast strain AM1411 and its derivatives were cultured in YEP-Lac liquid medium, diluted and plated on YEPD and YEP-Gal plates at ~50–100 cells per plate, and viability following the DSB was calculated as a ratio of cfu on YEP-Gal to YEPD. Colonies grown on YEP-Gal (and on YEPD as a control) were replica-pated onto SC-Leu, SC-Ade, and based on the phenotype were classified as follows: Leu+ Ade+ as GC, Leu- Ade+ as BIR or BIR associated with GCR, Leu- Ade- white as HC, and Leu- Ade- red as CL. The full analysis of DSB repair outcomes is presented in Supplementary Data 4. To distinguish BIR from GCR outcomes, DNA purified from Leu- Ade+ repair outcomes was analyzed by CHEF, with initial switch time of 10 s, final switch time of 35 s, running time of 40 h, and voltage of 6 Volts/cm, similar to 85.

Kinetics of BIR synthesis
The kinetics of BIR synthesis was analyzed using our recently developed ddPCR-based method called Assay for Monitoring BIR Elongation Rate (AMBER) 49. Yeast cells were cultured overnight in 5 ml SC-Leu medium, then transferred to YEP medium containing 2% raffinose and incubated for approximately 16 h, until the cell density reached ~5×106 cells per ml. A 10 ml aliquot was collected for analysis of pre-DSB copy number (“0h”), and 2% galactose was added to the remaining culture to induce DSB formation. Samples (10 ml each) were taken every 2 h for up to 10 h. Cells were resuspended in spheroplasting buffer (0.4 M sorbitol, 0.4 M KCl, 40 mM sodium phosphate buffer [pH 7.2], 0.5 mM MgCl2) containing 20T zymolyase (250 µg per ml) and incubated at 37°C for 10 min to digest the cell wall. Approximately 2.5×105 cells were then centrifuged at 1000×g for 2 min, and the supernatant was discarded. The pellet was resuspended in 400 µl of 1× NEB rCutSmart buffer (B6004S). Next, 4 µl of 10% SDS was added, and the mixture was incubated at 60°C for 15 min. The sample was cooled on ice, followed by addition of 50 µl 10% Triton X-100. RNase A (20 µl, 10 mg per ml) was then added, and the mixture was incubated at 37°C for 30 min. Subsequently, 20 µl protease K (10 mg per ml) was added and incubated at 60°C for 30 min. After cooling on ice for 5 min, 0.5 ml of phenol–chloroform was added, and the mixture was centrifuged at 16,000×g for 15 min. The aqueous phase was extracted with an additional 0.5 ml of phenol–chloroform, mixed by inversion, and centrifuged. DNA was precipitated from the final aqueous phase by adding 35 µl of 3 M Sodium acetate [pH 5.2] and 0.35 ml of isopropanol, followed by incubation at room temperature for 30 min. DNA was pelleted by centrifugation at 16,000×g for 20 min, and the supernatant was removed. The pellet was washed with 0.5 ml 80% ethanol, centrifuged at 16,000×g for 5 min, and air-dried. The dried DNA pellet was resuspended in 100 µl of molecular-grade water for subsequent analysis. Purified DNA was quantified by ddPCR using standard reaction conditions. BIR synthesis was followed by tracking the copy number increase along the track of BIR normalized to copy number at ACT1 located on Chr VI, using QX600 ddPCR software, similarly to ref. 49. See Supplementary Data 5 for the sequences of oligos used for this assay. Also, see Supplementary Data 6 for AMBER data obtained in each individual experiment.

Genome-wide screen for BIR genes
The screen was performed by the following steps:
1. AM3874 (Supplementary Data 1) cells were cultured in 500 ml liquid YEPD medium for 24 h. Concomitantly, the cells from the MATα YKO yeast collection (horizondiscovery.com/en/non-mammalian-research-tools/products/yeast-knockout), which represents a collection of approximately 5000 strains with deletions of individual non-essential genes in the BY4742 strain with the genotype MATα his3Δ-1 leu2Δ0 lys2Δ0 ura3Δ0 were grown overnight in 96-well plates with 100 µL of liquid YEPD medium per well.
2. 100 µl of AM3874 culture was added to each well of the 96-well plates for mating with the individual gene deletion strains. The strain mixtures were incubated overnight to allow efficient strain hybridization.
3. To select for the resulting hybrids (diploids), 10 µl of each mixture was placed on YEPD plates containing G418 (0.5 g per liter) and nourseothricin (0.1 g per liter), followed by overnight incubation.
4. Plates from Step 3 were replica plated onto sporulation (Kac) medium and incubated for 72 h at 30°C.
5. The sporulating cultures were replica plated onto YEPD plates containing G418 (0.5 g per liter), nourseothricin (0.1 g per liter), and cycloheximide (0.01 g per liter) and incubated for 48 h at 30°C.
6. Plates were replica plated onto SC-(Leu, His, Ura) and incubated for 48 h at 30°C.
7. Plates were replica plated onto YEPD+G418, nourseothricin, and cycloheximide and incubated for 48 h at 30°C.
8. Plates were replica-plated to SC-Lys and incubated for 48 h at 30°C.
9. BIR-defective mutants were identified based on a reduced number of Lys+ papillae on the patch (semi-quantitative BIR efficiency assay) compared to WT.

Assessment of cell cycle progression by microscopic analysis
A BIR repair time-course experiment was conducted in derivatives of the AM1411 strain in YEP-Lac, similar to described in ref. 85. Fifty-ml cell samples were collected before and at time points following DSB induction (galactose addition) for 12 h. To fix live cells, a spun-down pellet was resuspended in 500 μL of water, which was then thoroughly mixed with 500 μL of 100% ethanol. The mixture was stored at 4°C until the entire time-course experiment was completed. Fixed cells were twice washed with sterile water. Prior to imaging, 20–50 μL of the mixture was aliquoted in a new microcentrifuge tube and spun down at 800×g for 1 min, and the supernatant was completely removed. The pellet was then resuspended in 12 μL SlowFade TM DAPI (Thermo Fisher #S36964) and used to prepare microscope slides. Images were taken using Leica Widefield fluorescence microscope with a 40× magnification lens and acquired using LASX software.

ChIP assay
Yeast cells were cultured in YEP-raffinose liquid medium overnight at 30°C to achieve a density of 1×107 cells/mL before adding galactose (2% final concentration) to introduce DSB. Cells were fixed in formaldehyde (1% final concentration) to cross-link DNA–protein complexes. After 10 min, the cross-linking reactions were quenched by the addition of glycine (125 mM), and the reaction continued for 5 min on a shaker. The cells were collected by centrifugation and washed first with TBS (125 mM Tris-HCl [pH 7.4], 130 mM NaCl, 5 mM KCl, 0.9 mM CaCl2, and 0.5 mM MgCl2) supplemented with glycine (125 mM), followed by a second wash in TBS alone. Cells were resuspended in lysis buffer (50 mM HEPES [pH 7.5], 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mg/mL bacitracin, 1 mM benzamidine, and 1 mM PMSF). Glass beads were added to the cells suspension, followed by cell disruption in a bead beater for 6 min (1 min disruption each time with 2 min on-ice intervals). The cell lysate containing the genomic DNA was sheared to fragments with an average size of 0.5 kb by sonication (Bioruptor® Pico Sonicator B01080010). The cell debris was sedimented by centrifugation at 16,000×g for 20 min, and the collected supernatant was distributed into IP (Input) and Non-IP (Non-Input). For the IP samples, incubation was performed overnight with anti-c-Myc antibody (Sigma M4439) or anti-Rad51 antibody (a gift from Dr. Patrick Sung) at 4°C, followed by the addition of Dynabeads Protein G (Thermo, 1004D) and continued incubation for 4 h. Beads were then washed twice with lysis buffer, twice with high-salt lysis buffer (lysis buffer with 500 mM NaCl), twice with wash buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 250 mM LiCl, 0.5% sodium deoxycholate, and 0.5% Igepal CA-630), and twice with 1× TE. DNA–protein complexes were eluted by incubating beads with 100 μL of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, and 1% SDS) for 30 min at 65°C. Reverse cross-linking was carried out overnight at 65°C, followed by proteinase K treatment and phenol–chloroform purification of DNA. Purified DNA was analyzed by qPCR using the PowerUp SYBR Green Master Mix (Applied Biosystems, 4367659). Enrichment was normalized to the ACT1 locus. Accumulation of targeted proteins after DSB measured by ChIP is calculated by subtraction from before DSB.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Liu et al. conducted a genome-wide screen to identify new break-induced replication (BIR) genes in yeast using an ectopic BIR assay with non-essential gene knockouts (KOs). This screen uncovered 37 novel BIR genes involved in various biological processes, including the spindle assembly checkpoint (SAC), the spindle position checkpoint (SPOC), nuclear pore/migration, and chromatin and post-translational modifications.\nThe study employs multiple assays, including another BIR assay (disomic assay) and the AMBER assay, to further validate and analyze the potential roles of SAC and SPOC in BIR. The authors also discuss the possible influence of epigenetic modifications of chromatin and nuclear pore components; however, these sections lack a clear mechanistic explanation, making them feel incomplete. It is somewhat surprising that so many pathways are interconnected with BIR, and many likely influence BIR indirectly by broadly regulating the cell cycle and stress responses. Nonetheless, the study is rigorous, employing multiple validation assays, and provides an initial framework for further exploration of this critical process in both yeast and human cells.\nKey Questions and Considerations:\n1. Can we test whether the prolonged duration of the current BIR system is responsible for its dependence on SAC? If we analyze BIR systems that require shorter DNA synthesis, as described by Symington or Haber, would BIR still be dependent on SAC or SPOC?\n2. Can we distinguish whether the authors observed low cell viability or simply a prolonged cell cycle delay due to extended BIR in SPOC mutants?\n3. Is the lethality of nup188 not offset by mad2 deletion because it may also be involved in the DNA damage response (DDR)? Is deletion of RAD9 partially rescue the low viability in nup188?\n4. The discrepancy between BIR defects in mad2 using the disomic versus ectopic reporters is puzzling. The authors should resolve this inconsistency and ensure that candidate mutants showing BIR defects are consistently identified, regardless of the strain used to study BIR function.\n5. Why is half crossover (HC) formation reduced in bub3 mutants?\n6. The selection of 92 genes out of approximately 300 based solely on known gene function seems arbitrary. What criteria were used for this selection? The full list should be provided in the supplementary data.\n7. Besides EAF3, did the screen identify any other genes that enhance BIR?\n8. I noticed that HC formation is significantly increased in mad2 mutants. Can HC still generate Lys+ colonies in the ectopic assay?\n9. In bub2 mutants, HC is not detected. What could be the underlying reason?", "reviewer_2": "Hiu et al. conducted a genetic screen focused on non-essential genes in budding yeast to identify factors mediating break-induced replication (BIR), a DNA repair pathway that can drive chromosome instability and contribute to tumorigenesis in mammals. The results present a list of 37 genes as novel candidate regulators of BIR-mediated repair. Most of the gene hits are known to be associated with or linked to diverse molecular pathways. The authors assessed the impact of many of these hits in various versions of BIR reporter assays, which included different genetic reporter assays and copy-number assessments targeting chromosomal regions proximal or distal to the DNA break site (serving as a readout of early vs. late BIR; or BIR initiation vs. BIR progression). Experiments involved evaluating the usage of BIR compared to other potential options (e.g., GC, HC, or CL), cell cycle progression, and cell viability. Overall, the authors draw several main conclusions: the Spindle Assembly Checkpoint (SAC) is required for BIR, the Spindle Position Checkpoint (SPOC) is necessary for BIR, Nuclear pore proteins (Nup84 vs. Nup188) have multiple roles in BIR, and various post-translational modifications are important for successful BIR repair.\nOverall, the genetic screening conducted will interest researchers across various fields, including yeast genetics, checkpoint signaling, and genome stability. The authors deserve commendation for utilizing and clearly explaining several sophisticated genetic assays to evaluate BIR within different chromosomal contexts. However, this reviewer has several significant concerns that prevent the publication of this manuscript in its current form.\nMajor concerns:\nThe study is too preliminary and underdeveloped. In its present form, it may be better suited for a journal that reports genetic screen results without a significant level of mechanistic understanding or conceptual advance. However, as outlined below, these weaknesses may be addressable through a major revision.\nMany unsubstantiated assumptions and speculations are made, leaving the data insufficient to support the claims. A significant weakness of the study is the interpretation of weak correlations as causations. The authors often conflate the effect of knocking out individual genes with the disruption of the known pathway where the gene product typically operates, and no robust experiments or controls are executed to establish strong causal relationships between the allegedly impacted pathway and the observed decrease in BIR.\nAdditionally, within each pathway claimed to be required for efficient BIR, how many non-essential genes exist in yeast, how many of those genes were identified as hits in the genetic screen, and how many have been evaluated in post-screen BIR validation assays? In other words, what percentage of non-essential genes linked to SAC, SPOC, NPCs, etc., are required for efficient BIR? Is the relationship between each of these sets of genes epistatic (e.g., BUB1, BUB3, and MAD2; Nup84 and Nup188; etc.) in terms of their effect on BIR?\nHow do the authors rule out the possibility that the proposed genes may impact BIR by operating independently of their known functions, such as through moonlighting-type roles? Similarly, additional molecular or cellular biological mechanistic insight would be beneficial. For instance, what is the exact molecular relationship between factors that regulate the proper attachment of microtubules to the kinetochore during mitosis and the supposedly prolonged G2/M arrest required for BIR repair? Which molecular steps of BIR-based repair rely on Nup84 versus Nup188, and why? How does this explain the apparent dependence on Nup84 for BIR initiation and on Nup188 for BIR completion? Overall, the authors could focus on providing further mechanistic insights related to a few of their numerous major claims to offer more robust support while possibly toning down other claims that lack detailed mechanistic/experimental insight.\nStatistical analyses were often not depicted in the graphs (e.g., Fig. 1b, 2a,e-h, 3c-k, 4a-c,f-g, 5a-b,g-j, 6a,d-h) or may have been inappropriate. For instance, the supplemental table with the original results of the BIR screen (Supp Table 2) includes a t-test comparing all mutants to the WT. Since all mutants are compared to the same WT condition, the t-test is likely inappropriate and should be replaced with an ANOVA using multiple comparisons or a similar test that adjusts for multiple comparisons. Whether and how correcting the statistical analyses would change the list of screen hits identified and the overall results is a significant question. Additionally, the statistical analyses presented were sometimes unclear; for example, in Fig. 2d, was a Bonferroni correction or a similar adjustment applied for the multiple comparisons involving the WT condition?\nCaution is advised when interpreting the knockout of a specific gene that affects BIR in only some of the BIR assays utilized. How might the specific chromosomal context of the reporter DSB assay influence the interpretation of these results?\nOther point(s):\nAlthough the text is well-written, a more thorough proofreading or professional English editing would significantly enhance the manuscript. A particularly frequent mistake was concerning the use of the definite article \"the.\"\nGiven the important concerns outlined above, the manuscript's title and abstract could be misleading and should be revised with care.", "reviewer_3": "The manuscript by Liu, Lee et al represents a highly valuable addition to the understanding of the BIR process, a very important sub-pathway of homology-mediated repair of broken chromosomes. Defects in this process lead to the formation of structural genomic variants that are directly implicated in many disease processes. As noted by the authors, the number of cellular factors known to be involved in BIR is relatively limited, especially due to the lack of systematic efforts to identify them. This study is highly innovative in at least two ways. First, it reports on the results of such a systematic screen and uncovers dozens of new BIR factors, opening multiple exciting opportunities for follow-up research. Second, the authors present a comprehensive characterization of some of the most intriguing hits from their screen, in particular the genes involved in late cell cycle checkpoints (SAC and SPOC). The latter is very helpful in clarifying the relationship between the slow kinetics of BIR repair, and the need for fast-dividing cells to delay mitosis until BIR is completed. The authors also provide initial investigations of hits associated with the nuclear pore complex and chromatin structure, although not quite as in depth. Until now, studies of the BIR mechanism mostly focused on factors directly involved in DNA metabolism and repair (eg Pol32, Pif1, Rad51). Following this study, the range of cellular functions that will be investigated is expected to expand greatly, thus representing an influential contribution to the field.\nBelow are relatively minor comments, curiosities, suggestions that the authors and editors might wish to consider in a revised manuscript.\n-The authors did a genomic (CHEF) analysis (Fig. 5) of the Leu- Ade+ clones derived from the nup mutants and showed that some of them were not regular BIR, but instead contained some GCRs. That analysis was quite informative, thus raising the question: Was this type of genomic analysis also done for the bub mutants (Fig. 2,3,4)? If not, should it be done? Given that the SAC and SPOC pathways were the major focus of the work following the screening phase, I think it might be important to add some information about this. In other words, how sure are the authors that Leu- Ade+ clones derived mutants such as bub2D or bfa1D actually mostly carry BIR products?\n-Another aspect that I think should be commented on is the remarkable absence of half-crossovers (HC) in bub2D and nup184D. Both of these mutants displayed a massive drop in viability following DSB induction, and in both HCs were almost entirely gone. Is there a relationship? The authors included some discussion about gene conversions (GC), pointing out that GCs are in a “fast” repair class of products. HCs might also be in that “fast” repair group, so why are HCs gone while GCs increase (in bub2D)? I think this warrants some discussion.\n-Finally, and not as relevant, I was a bit intrigued by the meaning of flower and bee in Figure 1e. I am not against it, but I did not find any comment in the manuscript or the figure legend to explain its meaning. I suggest the authors add some comment to provide context to the readers about why the diagram was drawn as such."}, "author_response": "Response to reviewer’s comments:\nReviewer #1\nReviewer 1, Comment 1. Liu et al. conducted a genome-wide screen to identify new\nbreak-induced replication (BIR) genes in yeast using an ectopic BIR assay with non-\nessential gene knockouts (KOs). This screen uncovered 37 novel BIR genes involved in\nvarious biological processes, including the spindle assembly checkpoint (SAC), the\nspindle position checkpoint (SPOC), nuclear pore/migration, and chromatin and post-\ntranslational modifications.\nThe study employs multiple assays, including another BIR assay (disomic assay) and\nthe AMBER assay, to further validate and analyze the potential roles of SAC and SPOC\nin BIR. The authors also discuss the possible influence of epigenetic modifications of\nchromatin and nuclear pore components; however, these sections lack a clear\nmechanistic explanation, making them feel incomplete. It is somewhat surprising that so\nmany pathways are interconnected with BIR, and many likely influence BIR indirectly by\nbroadly regulating the cell cycle and stress responses. Nonetheless, the study is\nrigorous, employing multiple validation assays, and provides an initial framework for\nfurther exploration of this critical process in both yeast and human cells.\nReviewer 1, Response 1. We thank the Reviewer for these comments. Indeed, our\ngoal was to create a complete list of non-essential yeast genes that are required for\nBIR. As the Reviewer notes, this effort yielded a long list of genes across many\npathways, which made it impossible for us to complete mechanistic analyses for every\ngene/pathway. Instead, we decided to prioritize depth over breadth regarding\nmechanistic studies, selecting only a few pathways for characterization. In our revised\nmanuscript, we maintained our focus on these selected pathways, but we have\nsignificantly expanded the amount of mechanistic detail regarding their involvement in\nBIR.\nReviewer 1, Comment 2. Can we test whether the prolonged duration of the current\nBIR system is responsible for its dependence on SAC? If we analyze BIR systems that\nrequire shorter DNA synthesis, as described by Symington or Haber, would BIR still be\ndependent on SAC or SPOC?\nReviewer 1, Response 2. In response to this comment, we followed the Reviewer’s\nsuggestion and studied the dependence of SAC and SPOC genes in a yeast\nexperimental BIR system that requires shorter DNA synthesis, as published in (JAIN et\nal. 2009). DSBs in this system are induced at Chr V, and the centromere-proximal end\ninvades into Chr III to complete BIR repair by synthesizing 41 kb of DNA (Fig. 2b;\nSupplementary Fig. 3a, b; Supplementary Table 2.2). In this system (YSJ131), both\nmad2Δ (SAC) and bub2Δ (SPOC) significantly reduced BIR efficiency, which led to\nsignificantly reduced cell viability following DSB induction compared to wild type cells\n(Fig. 2b, Supplementary Fig. 3b). Therefore, this represents the third system in which\nwe demonstrate the dependence of BIR on the SAC and SPOC. However, to the\nReviewer’s point, we cannot fully exclude that this dependence could be decreased or\neliminated in some other exceptionally fast and efficient BIR systems.\nthe total amount of time the cell needs to finish repair, including time to identify a\nsuitable homologous sequence and to subsequently synthesize DNA. This notion is\nsupported by our finding that mad2Δ and bub2Δ were defective in gene conversion\n(GC) requiring 5 kb of DNA repair synthesis in an isogenic to YSJ131, system (YSJ133)\n(JAIN et al. 2009), in which the DSB is induced at the same location as in this new\nectopic BIR system (YSJ131) (Supplementary Fig. 3a, b); however, mad2Δ and bub2Δ\nwere as proficient as WT at completing GC with copying ~5 kb in our allelic disomic\nsystem in which the homology search between allelic locations is much faster (Fig. 2d,\nFig. 4d). The new data were added to Fig. 2b and to the Supplementary Fig. 3a-b, with\nall raw data included in Supplementary Tables 2.2 and 2.3. Also, we added the following\nsentences to the Results section:\n• In support of this hypothesis, we observed a defective BIR in bub1Δ, bub3Δ, and\nmad2Δ mutants in two ectopic BIR systems (AM3422 and YSJ131; Fig. 2 a, b;\nSupplementary Fig. 3a, b, Supplementary Tables 2.1 and 2.2).\n“However, in an ectopic system (YSJ133), where the homology search takes\nlonger and even GC can triggers checkpoint arrest BUB2 deletion led to a\nmodest decrease in GC efficiency (Supplementary Fig. 3b), but the defect of BIR\nin this system (YSJ131) was more severe (Supplementary Fig. 3b).”\nReviewer 1, Comment 3. Can we distinguish whether the authors observed low cell\nviability or simply a prolonged cell cycle delay due to extended BIR in SPOC mutants?\nReviewer 1, Response 3. To address this concern, we plated bfa1Δ and bub2Δ cells\non YEPD and YEP-Gal media and incubated the plates at 30 οC for 10 days. Cell\nviability was calculated as the number of colonies formed on YEP-Gal over the number\nof colonies formed on YEPD. Viability in bfa1Δ and bub2Δ cells after 10 days was low\nand similar to viability previously observed after 5 days of growth. Importantly,\nmicroscopic examination demonstrated that, among the cells that did not form colonies,\n81% of bfa1Δ (34/42) and 47% of bub2Δ (26/55) cells stayed below the 4-cell stage.\nAmong the other plated cells that didn’t form colony, most divided fewer than 3 times,\nand all of them contained fewer than 14 cells. Our results indicate that the low colony-\nforming efficiency we observed following DSB induction in SPOC mutants results from\ncell death rather than from slow growth associated with a prolonged cell cycle delay.\nThese data were added to Supplemental Fig. 3d, and the following sentence was added\nto the Results section:\n• “Microscopic examination demonstrated that, following plating on YEP-Gal\nmedium, most bub2Δ cells divided no more than twice, and no cells formed\nmicro-colonies after DSB induction (Supplementary Fig. 3d) confirming lethality\nfollowing the DSB.”\nReviewer 1, Comment 4. Is the lethality of nup188 not offset by mad2 deletion\nbecause it may also be involved in the DNA damage response (DDR)? Is deletion of\nRAD9 partially rescue the low viability in nup188?\nadditional experiments in nup188Δmad2Δ mutants of the disomic BIR strain. These\nadditional data allowed us to conclude that, following DSB induction, cell viability in the\ndouble mutant is slightly but significantly increased as compared to nup188Δ alone (Fig.\n5a).\nNext, based on the Reviewer’s suggestion, we generated a nup188Δrad9Δ double\nmutant in our chromosome III disomic BIR strain. After DSB induction in this strain, we\nobserved that RAD9 deletion partially rescued cell viability in the nup188Δ context (Fig.\n5a). Unfortunately, we were unable to analyze the efficiency of BIR in\nnup188Δmad2Δrad9Δ triple mutants because of their highly unstable (always\naneuploid) genomes. Collectively, our results lead us to propose that Nup188 can\nparticipate in BIR through the interaction with DDC and SAC pathways.\nThese data were added to Fig. 5a-b, and the raw data were included in Supplementary\nTable 4. In addition, we added the following sentence to the Results section:\n• “Additional support came from the observation that deleting MAD2 or RAD9 in\nnup188Δ significantly increased viability after DSB induction, albeit BIR remained\ndefective (based on the distribution of repair outcomes) (Fig. 5a-b,\nSupplementary Fig. 6b, e, f).”\nReviewer 1, Comment 5. The discrepancy between BIR defects in mad2 using the\ndisomic versus ectopic reporters is puzzling. The authors should resolve this\ninconsistency and ensure that candidate mutants showing BIR defects are consistently\nidentified, regardless of the strain used to study BIR function.\nReviewer 1, Response 5. To address this concern, we conducted several additional\nbiological repeats of the BIR assay in mad2Δ mutants of the ectopic BIR system\n(AM3422). This allowed us to determine that MAD2 deletion mildly but significantly (by\nindividual Welch’s t-test) decreased BIR efficiency from 42% in WT to 37% in mad2Δ\n(Fig. 2a), even though the one-way ANOVA test did not show significance\n(Supplementary Table 2.1). Importantly, we also observed decreased BIR efficiency in\nmad2Δ in an additional ectopic BIR system (YSJ131) by both Welch’s t-test and by\nOne-way ANOVA (Fig. 2b; Supplementary Table 2.2; see our response #2). In addition,\nmad2Δ significantly decreased BIR in allelic BIR system (Fig. 2d). Thus, mad2Δ\nsignificantly decreased BIR efficiency in two BIR systems, and the data were borderline\nsignificant for the third system. We believe this borderline result could be explained by\nour inability to distinguish BIR and half-crossovers in this ectopic system as Reviewer 1\nsuggested in comment 9.\nReviewer 1, Comment 6. Why is half crossover (HC) formation reduced in bub3\nmutants?\nReviewer 1, Response 6. To address this, we performed several additional biological\nrepeats of these experiments, the results of which confirmed that the frequency of HCs\nin bub3Δ is slightly but significantly lower compared to WT (Fig. 2d). We do not currently\nhave a clear explanation for this phenomenon. This lower frequency in bub3Δ could, in\n3\nprinciple, reflect the way that intermediates of unfinished BIR are resolved in these\nmutants. We acknowledge that, while resolution of these intermediates can result in HC\nformation, it can also lead to formation of outcomes indistinguishable from chromosome\nloss (CL). It is also possible that HC events could undergo secondary strand invasion\nand produce HC cascade events that would be phenotypically indistinguishable from\nBIR. With our current data, it is hard to distinguish among these possibilities. Our results\nin Fig. 2d have been updated with our new data.\nReviewer 1, Comment 7. The selection of 92 genes out of approximately 300 based\nsolely on known gene function seems arbitrary. What criteria were used for this\nselection? The full list should be provided in the supplementary data.\nReviewer 1, Response 7. Out of 335 candidate BIR genes identified in our screen,\n243 genes were excluded from further investigation. The list of excluded genes\nincludes:\n1) 28 genes that were known to be false positives, including:\na. 10 genes located on chromosome V between CAN1 and the telomere,\nwhich appeared as false positives on our candidate gene list because\nselection of mutant of these genes will against the selection of the BIR\nchromosome.\nb. 18 genes involved in lysine biosynthesis, which represent false positives\nbecause our BIR assay was based on Lys+ selection.\n2) 19 BIR genes that were previously described, and, therefore, deprioritized for our\ncurrent study.\n3) Because our study aimed to identify genes directly involved in BIR occurring\nwithin the nucleus, we excluded 193 genes that have no known role in DNA\nmetabolism, including:\na. 132 genes with mitochondrial or metabolism/biosynthetic roles,\nb. 28 genes involved in transcription or RNA metabolism,\nc. 20 genes that control ribosome- or endoplasmic reticulum-related\nfunctions, and\nd. 13 genes with unknown functions\ne. 3 other genes involved in cell cycle regulation\nIn response to this Reviewer’s comment, the full list of genes identified from the screen\nand whether they were included in or excluded from further analysis was incorporated\ninto Supplementary Table 3, with the reasoning behind the exclusion of some genes\nincluded in the table legend. In addition, to clarify the criteria used to select 92 genes for\nfurther analysis, the Results section was corrected as follows:\n• “We identified 335 KO strains (Supplementary Table 3) with fewer Lys+ papillae\ncompared to WT, 19 of which (e.g. RAD52, RAD51, POL32, SRS2, RAD54;)\nwere already known to promote BIR. From the rest, 92 genes were selected for\nfurther characterization based on their known roles in processes related to DNA\nmetabolism, including, for example spindle checkpoints, nucleopore structure,\nand post-translational modification of proteins associated with DNA\n4\n(Supplementary Table 3.1). The other identified genes, including mitochondrial\nand metabolism/biosynthetic genes (Supplementary Table 3.2), were expected to\naffect BIR indirectly, and, therefore, were not prioritized in this study. “\nReviewer 1, Comment 8. Besides EAF3, did the screen identify any other genes that\nenhance BIR?\nReviewer 1, Response 8. Yes, our screen identified 6 genes for which BIR efficiency\nwas increased upon their deletion: UBA3, UBA4, MUB1, CCS1, YVH1, and PIB2\n(Supplementary Table 2.1 and 2.3). To address this Reviewer’s concern, these genes\nare listed in the Results section:\n• “Interestingly, deletions of the remaining 6 genes (UBA3, UBA4, MUB1, CCS1,\nYVH1, and PIB21) resulted in increased BIR efficiency, suggesting these genes\nrepresent BIR suppressors; however, these genes were probably selected by our\nscreen due to conferring a slow-growth phenotype (Supplementary Table 3).”\nReviewer 1, Comment 9. I noticed that HC formation is significantly increased in\nmad2 mutants. Can HC still generate Lys+ colonies in the ectopic assay?\nReviewer 1, Response 9. We thank the Reviewer for this comment. We agree that,\nbecause all BIR occurs during G2 (a two-chromatid stage of the cell cycle), an HC that\nformed in our ectopic system followed by co-segregation of an HC product with a\nsecond copy of an intact donor would produce an outcome indistinguishable from BIR.\nWhile, in the allelic BIR system, HCs (at least a half of them) can be readily\ndistinguished from BIR, no such distinction can be made in the ectopic BIR system.\nWe are not sure why mad2Δ leads to a small but significant increase in HCs, but this\nobservation may explain why it was so difficult to detect the decrease in BIR in mad2Δ\nmutants in the ectopic system; specifically, HCs were likely counted as BIR events in the\nectopic system.\nReviewer 1, Comment 10. In bub2 mutants, HC is not detected. What could be the\nunderlying reason?\nReviewer 1, Response 10. We reported here that, in the absence of Bub2, the majority\nof cells undergoing BIR die due to the onset of cytokinesis in cells where nuclear\ndivision is delayed; only a very small portion of BIR outcomes—likely those that\ncomplete repair before the onset of dysregulated cytokinesis—form colonies. This result\npredicts that anything that delays or interrupts BIR would promote cell death in bub2∆\ncells. The formation of HCs is likely associated with delays during BIR repair, such as\npausing, interruption, resolution of BIR intermediates, or formation of a secondary DSB\n(breakage of the BIR donor chromosome), which could extend nuclear division arrest.\nTherefore, it is expected that no HC events would complete before the onset of\ncytokinesis, because any bub2∆ cells in the process of forming an HC would have\nextended nuclear arrest resulting in cell death. To address this comment in the revised\nmanuscript, we included the following sentence in the Results section:\n5\n• “The fraction of HC outcomes among survivors was very low in bub2Δ and bfa1Δ\nmutants (Fig. 4d), likely because of the extended time required for HC formation,\nwhich involves secondary breakage of the donor chromosome. Collectively, these\nresults support that the viability of cells undergoing BIR depends on the SPOC.\nReviewer #2 (Remarks to the Author):\nReviewer 2, Comment 11. Many unsubstantiated assumptions and speculations are\nmade, leaving the data insufficient to support the claims. A significant weakness of the\nstudy is the interpretation of weak correlations as causations. The authors often conflate\nthe effect of knocking out individual genes with the disruption of the known pathway\nwhere the gene product typically operates, and no robust experiments or controls are\nexecuted to establish strong causal relationships between the allegedly impacted\npathway and the observed decrease in BIR.\nReviewer 2, Response 11. Our study reports a genome-wide screen wherein we\nidentified 335 gene candidates involved in BIR, and we confirmed the roles of 30\ngenes—grouped into 5 functional groups/pathways according to their known functions—\nin BIR using multiple BIR experimental systems. Because of the scope of our findings, it\nwas necessary to prioritize particular genes, which we did based on our expectation that\ncertain genes might be more likely to uncover clear molecular pathways that contribute\nto the efficiency of BIR. To address this Reviewer’s concern, we performed additional\nexperiments to elucidate the role of three of the pathways we identified in this study: (i)\nthe SAC pathway, ii) the SPOC pathway, and (iii) the nucleopore complex. The following\nnew experiments were conducted:\n1. We conducted a series of new cytological experiments to analyze BIR repair in\ntwo additional SAC mutant lines: bfa1Δ and bub2Δmad2Δ. These experiments\ndemonstrated that, similarly to bub2Δ, only a small fraction (7%) of bfa1Δ cells\nsuccessfully formed colonies (Fig. 4c, Supplementary Table 4), while the vast\nmajority of cells formed cell chains with anucleate cells (Fig. 4f, g, j; see\nSupplementary Table 7 for all raw data), indicative of cytokinesis onset prior to\nnuclear division and BIR completion. In bub2Δmad2Δ, the fraction of anucleate\ncells forming chains was decreased compared to bub2Δ (Fig. 4h, j; see\nSupplementary Table 7 for all raw data), and a significant fraction of bub2Δmad2Δ\ncells demonstrated earlier recovery from nuclear checkpoint arrest due to\nabsence of the SAC consistent with the increased viability of the double mutant as\ncompared to bub2Δ.\n2. We performed an epistasis analysis involving SAC and SPOC mutants and\ndetermined the effects of double mutations on BIR efficiency. These experiments\nshowed that the low viability of SPOC mutants after DSB induction could be\nrescued by deactivation of the SAC pathway. In particular, the viability of\nbub2Δmad2Δ cells was increased compared to bub2Δ cells (Fig. 4c,\nSupplementary Table 4), which supported our hypothesis that discoordination\nbetween nuclear division (following nuclear division arrest) and the onset of\ncytokinesis underlies the observed lethality in SPOC mutants after DSB induction.\nIn addition, we observed that BUB1 is epistatic to MAD2 with respect to BIR\n6\nefficiency (Fig. 2d; Supplementary Fig. 4a, h; Supplementary Table 4), consistent\nwith MAD2 and BUB1 functioning in the same (SAC) pathway during BIR.\n3. We performed several detailed analyses of the roles of Nup84 and Nup188 in\nBIR. (1) Using chromatin immunoprecipitation (ChIP), we now demonstrate that\nboth Nup84 and Nup188 localize to the DSB (Fig. 5g). (2) We also performed\nChIP using an anti-Rad51 antibody, which demonstrated that the absence of\nNup84 does not affect Rad51 loading at the DSB site of the broken (recipient)\nchromosome (Fig. 5h). Further, we observed that Rad51 loading on the donor\nchromosome (reflecting strand invasion) was reduced in nup84∆ compared to wild\ntype cells, however, a substantial level of Rad51 loading on the donor was\nobserved even in the absence of Nup84 (Fig. 5h). Given our observation that the\ninitiation of BIR synthesis was defective in nup84Δ mutants, we propose that\nNup84 facilitates strand invasion, and it plays a particularly important role after\nstrand invasion and before initiation of BIR synthesis. For example, Nup84 may\nbe important for the formation and stabilization (maturation) of the D-loop or\ninitiation of BIR synthesis (possibly by facilitating the recruitment of Rad54, PCNA,\nDNA polymerases, or Pif1 helicases). (4) We now demonstrate that nup84Δ is\nepistatic to nup188Δ with respect to BIR efficiency and cell viability after DSB\ninduction (Fig. 5a, b), supporting our model wherein Nup84 functions prior to\nNup188. (5) In addition, our genetic analysis of BIR efficiency and cell viability\nafter DSB induction in nup188Δmad2Δ and nup188Δrad9Δ double mutants\nsuggested a potential role for Nup188 in checkpoint control during DSB repair,\nwhich is a novel finding (Fig. 5a, b).\n4. We introduced deletions of 30 newly identified BIR genes in an additional BIR\nexperimental system (YSJ131; see our response #18) (Supplementary Table 2.2,\nand we tested the effects of several mutations in the GC ectopic system (see our\nresponse #2), which provided additional mechanistic insights (Supplementary\nTable 2.3).\nAll of these new results are included in the Results section of the revised manuscript\nand discussed in the revised Discussion section.\nReviewer 2, Comment 12. Additionally, within each pathway claimed to be required for\nefficient BIR, how many non-essential genes exist in yeast, how many of those genes\nwere identified as hits in the genetic screen, and how many have been evaluated in\npost-screen BIR validation assays? In other words, what percentage of non-essential\ngenes linked to SAC, SPOC, NPCs, etc., are required for efficient BIR?\nReviewer 2, Response 12. Based on the literature, there are 5 non-essential SAC\ngenes (HOYT et al. 1991; LI AND MURRAY 1991; WEISS AND WINEY 1996), and our screen\nsuccessfully identified two: BUB1 and BUB3. Further analysis revealed that deletion of\nMAD2 also reduced BIR efficiency from 42% in wild type cells to 37% (based on the\nindividual Welch’s t-test) in the ectopic BIR system (AM3422). This modest reduction\nmay explain why MAD2 was not identified in our initial screen (a discussion of this\nphenotype is provided in our response #9). Thus, the three main proteins known to\ncooperate to execute the SAC are all important for BIR. We also tested mad3Δ mutants,\nwhich showed no effect on BIR. Interestingly, deletion of MAD1 significantly increased\nBIR efficiency in our ectopic assay (Supplementary Table 2.1). It was previously\n7\ndemonstrated that Mad1 functions as a scaffold to recruit Mad2 to the kinetochore, but\nthe function of Mad2 in suppressing APC can be Mad1 independent. Therefore, it is\npossible that transducing signals to SAC during BIR bears some unique features that\nare not yet understood.\nFor the SPOC pathway, 7 of 10 genes are non-essential, as described in (CAYDASI et al.\n2010). Our study identified two of them, BUB2 and BFA1, as BIR genes (Fig. 4), In\naddition, we identified three genes involved in nuclear migration (Supplementary Fig.\n3f), including JNM1, as well as components of the dynein pathway (NUM1 and APR1)—\nboth of which are functionally related to the SPOC—and our post-screen BIR validation\nassays demonstrated that they were all involved in BIR.\nSimilarly, 17 out of 31 subunits of the nuclear pore complex (NPC) are non-\nessential(HAMED AND ANTONIN 2021). Our screen only identified NUP188—a non-\nessential gene—while our further experiments also confirmed Nup84 as an important\nBIR protein (Fig. 5).\nReviewer 2, Comment 13. Is the relationship between each of these sets of genes\nepistatic (e.g., BUB1, BUB3, and MAD2; Nup84 and Nup188; etc.) in terms of their\neffect on BIR?\nReviewer 2, Response 13. To address this question, we constructed bub1Δmad2Δ\ndouble mutants and tested BIR efficiency. The double mutant had a BIR defect that was\nsimilar to the defect observed in each of the single mutants, suggesting that BUB1 and\nMAD2 are epistatic and act in the same pathway during BIR (Fig. 2d; Supplementary\nFig. 4a, h; Supplementary Table 4).\nWe also generated a nup188Δnup84Δ double mutants, and we found that the double\nmutants had increased cell viability after DSB induction compared to nup188Δ single\nmutants (Fig. 5a). Together with our other genetic and kinetics studies, this result\nsupported our initial hypothesis that NUP188 and NUP84 operate at different stages of\nBIR, with Nup84 acting during earlier steps while Nup188 contributes to the successful\ncompletion of BIR.\nIn response to this comment, several figure panels were created or changed including\nFig. 2d, Fig. 5a,b, Supplementary Fig. 4a, h, and Supplementary Fig. 6b, g of the\nrevised manuscript, and the following sentences were added in the Results section:\n• “In addition, bub1∆mad2∆ double mutants showed a BIR defect similar to each\nsingle mutant (Fig. 2d; Supplementary Fig. 4a, h).”\n• “We found that nup188Δnup84Δ double mutants had higher viability than\nnup188∆ (78% vs 12%, respectively) (Fig. 5a) and an altered distribution of DSB\nrepair outcomes (Fig. 5b, Supplementary Fig. 6b, g), supporting our hypothesis\nthat Nup188 participates later than Nup84 in BIR.”\ngenes may impact BIR by operating independently of their known functions, such as\nthrough moonlighting-type roles?\nReviewer 2, Response 14. We acknowledge it is difficult to completely rule out that\nsome of our newly identified genes impact BIR independently of their known primary\nfunctions, especially when dealing with more than 30 new genes that we characterized.\nWhile it will take many more years and laboratories to delineate the precise molecular\nroles of each of our newly identified genes, we think that the following arguments can be\nmade:\n1. The strength of our study, in our opinion, is that we were able to discover the\nroles of several members of the same functional group within BIR. Examples\ninclude SAC, SPOC, NPC, and several chromatin remodeling proteins. When\nseveral proteins from a functional group are confirmed to affect BIR efficiency, we\nthink it provides support for the idea that the whole pathway participates in BIR,\nand this makes it more likely that the identified proteins are cooperating to exert\ntheir known related functions, rather than by moonlighting in BIR.\n2. The functions of the DDC, SAC, and SPOC in BIR are supported by various\nmethods, including genetic, molecular biological, and cytological experiments,\nwhich together support our hypotheses that the DDC and SAC are required to\nprevent nuclear divisions prior to BIR completion, while the SPOC is required to\nprevent premature cytokinesis before nuclear division. The phenotypes observed\nin our BIR studies (especially those observed cytologically) are consistent with\nthese pathways playing their main roles in cell cycle regulation. Genetic\ninteractions between mutants from these three groups also support this idea.\n3. Our data obtained for Nup84 and Nup188 also suggest a direct role of the NPC\nin facilitating DSB repair through BIR. Our new ChIP data demonstrate that these\nproteins directly interact with broken DNA, consistent with previous findings that\nDNA fragile sites relocalize to the nuclear pore during DNA replication/repair to\nensure accurate synthesis and genomic stability.\nReviewer 2, Comment 15. Similarly, additional molecular or cellular biological\nmechanistic insight would be beneficial. For instance, what is the exact molecular\nrelationship between factors that regulate the proper attachment of microtubules to the\nkinetochore during mitosis and the supposedly prolonged G2/M arrest required for BIR\nrepair?\nReviewer 2, Response 15.\nTo address this Reviewer’s question, we hypothesized that, similar to (DOTIWALA et al.\n2010; ZHOU et al. 2024), the initiation of a DSB at MAT should be followed by spreading\nof γH2Ax across a large chromosomal region and reach the centromere, which initiates\ndetachment of microtubules from the centromere. This was proposed to activate the\nSAC in response to the DSB (DOTIWALA et al. 2010; ZHOU et al. 2024). To test this, we\nintroduced the hta1-S129A and hta2∆ double mutations in our disomic system to\neliminate the ability of histone H2A to undergo γH2Ax modification. These mutations\n9\ncaused a significant but very mild BIR defect, which was much less severe than the\ndefects we observed in strains with BUB1, BUB3 or MAD2 deletions (Supplementary\nTable 4). Therefore, an alternative mechanism might activate the SAC during BIR. It is\ninteresting, in this regard, that our results described in response #12 demonstrated that,\nwhile Bub1, Bub3, and Mad2 were required for successful BIR, MAD1 deletion led to\nincreased BIR efficiency (Supplementary Table 2.1). Because Mad1 is known to function\nas a scaffold for the recruitment of Mad2 to the kinetochore in response to a lack of\nmicrotubule attachment, it is possible that transduction of the signal to the SAC is\nindependent of microtubule attachment to the kinetochore. To this end, we are now\nexamining whether SAC activation during BIR might be dependent on Tel1 and Mec1,\nbut independent of the kinetochore, which has been proposed previously (KIM AND\nBURKE 2008). Also, we are exploring the interplay between DSBs at MAT and\ndetachment of microtubes from the centromere using a combination of genetic and\nmolecular biological approaches. All these experiments are expected to take a\nsignificant amount of time and will be described in a future publication.\nTo address this issue in the revised manuscript, we added the following statement to the\nDiscussion section:\n“For unrepairable breaks, it was established that activation of the SAC proceeds by the\nspreading of γH2AX from the DSB to the centromere on the broken chromosome,\ncausing kinetochore dysfunction and prolonging G2/M arrest. However, we observed\nthat in our allelic BIR system, an H2A-S129A mutant defective for γH2AX modification\nexhibited only a minor BIR defect, which was much less severe than the defect\nobserved in SAC mutants (Supplementary Table 4). Therefore, an alternative\nmechanism may activate the SAC during BIR. For example, previous studies have\ndemonstrated that SAC activation could be achieved in a Tel1- and Mec1-dependent but\nkinetochore-independent manner.\"\nReviewer 2, Comment 16. Which molecular steps of BIR-based repair rely on Nup84\nversus Nup188, and why? How does this explain the apparent dependence on Nup84\nfor BIR initiation and on Nup188 for BIR completion? Overall, the authors could focus on\nproviding further mechanistic insights related to a few of their numerous major claims to\noffer more robust support while possibly toning down other claims that lack detailed\nmechanistic/experimental insight.\nReviewer 2, Response 16. To address this question, we conducted ChIP, which\nshowed that both Nup84 and Nup188 participate in DSB repair by physically interacting\nwith broken DNA ends (Fig. 5g). We also found that Nup84 and Nup188 act sequentially\nduring BIR, with Nup84 participating in earlier steps and Nup188 near the end. In\naddition, we used the AMBER methodology, which showed no initiation of BIR synthesis\nin the absence of Nup84 (Fig. 5e; Supplementary Fig. 6h). Moreover, Rad51 ChIP in\nnup84∆ cells demonstrated wild-type levels of Rad51 loaded onto the broken\nchromosome, and a decreased but still substantial amount of Rad51 loaded on the\ndonor chromosome (Fig. 5h). This suggests that the absence of Nup84 did not affect\nDSB resection but partially impaired strand invasion and results in a critical defect in the\nstep following strand invasion. Altogether, our new data allowed us to pinpoint when\nNup84 acts during BIR: we now think that it helps during strand invasion and also plays\nan essential role after strand invasion and prior to the initiation of BIR synthesis. From\n10\nthese data, we propose that Nup84 could be important for recruiting BIR proteins, such\nas Rad54, Pif1, PCNA, or DNA polymerases, during D-loop stabilization and initiation of\nBIR synthesis.\nFurther, our new data support that Nup188 acts later during BIR than Nup84.\nFirst,\nAMBER demonstrated that, in the absence of Nup188, BIR synthesis can initiate but\nbecomes very defective towards its end (Fig. 5f; Supplementary Fig. 6h). Second,\ndeleting NUP84 in nup188Δ cells restored viability after DSB induction (Fig. 5a),\nwhich\nsuggested that the cell death observed in nup188Δ mutants resulted from formation of\nBIR intermediates mediated by Nup84. In addition, previous studies reported that\nhNUP188 interacts with the SAC and plays a role in regulating centrosome formation,\nindicating that yeast Nup188 could also play a role in checkpoint establishment during\nBIR. In support of this, our new genetic data demonstrate that inactivation of the SAC or\nDDC partially rescues cell viability following DSB induction in nup188Δ (Fig. 5a).\nIn response to this comment, we added new genetics data to Figures 5a and 5b and\nChIP data to Figures 5g and 5h. We also re-wrote the entire sections in the Results and\nDiscussion dedicated to nuclear pore proteins.\nReviewer 2, Comment 17. Statistical analyses were often not depicted in the graphs\n(e.g., Fig. 1b, 2a,e-h, 3c-k, 4a-c,f-g, 5a-b,g-j, 6a,d-h) or may have been inappropriate.\nFor instance, the supplemental table with the original results of the BIR screen (Supp\nTable 2) includes a t-test comparing all mutants to the WT. Since all mutants are\ncompared to the same WT condition, the t-test is likely inappropriate and should be\nreplaced with an ANOVA using multiple comparisons or a similar test that adjusts for\nmultiple comparisons. Whether and how correcting the statistical analyses would\nchange the list of screen hits identified and the overall results is a significant question.\nAdditionally, the statistical analyses presented were sometimes unclear; for example, in\nFig. 2d, was a Bonferroni correction or a similar adjustment applied for the multiple\ncomparisons involving the WT condition?\nReviewer 2, Response 17. In response to this comment, we took the following steps.\nFirst, we ensured that every figure and legend (including those indicated by the\nreviewer) contains details of the statistical analyses.\nSecond, we updated the statistical methods to account for multiple hypothesis testing,\nas suggested. Specifically, for the viability assays we now use one-way ANOVA (with\nDunnett’s post-hoc test, grouping all mutants tested in each system). We included the\nP-values from both the one-way ANOVA and the Welch’s t-test in Supplementary\nTables 2.1 and 2.2. For the BIR repair distribution assays, we applied the Benjamini–\nHochberg correction (with a false discovery rate of 0.05, grouping all mutants tested in\neach system) to the existing Chi-square test.\nImportantly, the vast majority of mutants that were previously shown to have significant\ndefects remained significantly defective based on the results obtained using these\nupdated statistical methods. For several mutants that were only nominally significant\n(significant before correction but not after), we updated the figures, tables, and text to\n11\ncorrectly reflect their defectiveness. Two of these mutants (bub1Δ and mad2Δ) remain\non the list of our BIR players as they were defective in 2 of our 3 BIR systems (YSJ131\nand AM1411) for plating outcomes. In addition, their BIR defect is supported by our\nddPCR-based AMBER method and by cytological studies. Thus, we believe it is\nessential to continue research even on mutants demonstrating modest defects by\nplating genetics, as they may uncover important mechanistic insights during the process\nof BIR and DNA repair.\nReviewer 2, Comment 18. Caution is advised when interpreting the knockout of a\nspecific gene that affects BIR in only some of the BIR assays utilized. How might the\nspecific chromosomal context of the reporter DSB assay influence the interpretation of\nthese results?\nReviewer 2, Response 18. In our original manuscript, we verified 33 individual BIR\ncandidate genes in our ectopic BIR strain (AM3422), and we also deleted many of these\ngenes in the disomic strain (allelic BIR system, AM1411) (Supplementary Table 4).\nThese BIR systems differ in several ways. First, in both systems, DSBs are induced in\ndifferent chromosomes and repaired by ectopic BIR in the first system and by allelic BIR\nin the second one. In response to this reviewer’s question, we introduced deletions of\n24 genes into a third, ectopic BIR system (YSJ131), which we received from Dr. James\nHaber (Supplementary Table 2.2). This BIR system was constructed in a different\ngenetic background and repairs using a different locus compared to the other two BIR\nsystems. Thus, we can now support our conclusions regarding the roles of newly\nidentified genes in BIR with data from three different BIR systems.\nThe results of our new experiments are included in Supplementary Table 2.2, and we\nadded the following sentence to our Results section:\n“In addition, we were able to introduce 24 of these 30 deletions in another ectopic\nBIR system (YSJ131), and each of these single-gene mutants showed BIR\ndefect;); in this system, the HO-induced break occurs in chromosome V, and BIR\nrepair is initiated by invasion into leu2 on chromosome III, followed by 41 kb of\nrepair synthesis 45 (Supplementary Fig. 3a, Supplementary Table 2.2).”\nReviewer 2, Comment 19. Although the text is well-written, a more thorough\nproofreading or professional English editing would significantly enhance the manuscript.\nA particularly frequent mistake was concerning the use of the definite article \"the.\"\nReviewer 2, Response 19. We made multiple changes to the text, and we enlisted the\nhelp of a professional scientific editor to review the entire manuscript. It is our hope that\nthis effort improved the readability of our revised manuscript.\nReviewer 2, Comment 20. Given the important concerns outlined above, the\nmanuscript's title and abstract could be misleading and should be revised with care.\nReviewer 2, Response 20. We thank the Reviewer for this suggestion. In response, we\nrevised the abstract and the title, and we hope the Reviewer finds these changes\nresponsive to their concern.\nthe understanding of the BIR process, a very important sub-pathway of homology-\nmediated repair of broken chromosomes. Defects in this process lead to the formation\nof structural genomic variants that are directly implicated in many disease processes.\nAs noted by the authors, the number of cellular factors known to be involved in BIR is\nrelatively limited, especially due to the lack of systematic efforts to identify them. This\nstudy is highly innovative in at least two ways. First, it reports on the results of such a\nsystematic screen and uncovers dozens of new BIR factors, opening multiple exciting\nopportunities for follow-up research. Second, the authors present a comprehensive\ncharacterization of some of the most intriguing hits from their screen, in particular the\ngenes involved in late cell cycle checkpoints (SAC and SPOC). The latter is very helpful\nin clarifying the relationship between the slow kinetics of BIR repair, and the need for\nfast-dividing cells to delay mitosis until BIR is completed. The authors also provide initial\ninvestigations of hits associated with the nuclear pore complex and chromatin structure,\nalthough not quite as in depth. Until now, studies of the BIR mechanism mostly focused\non factors directly involved in DNA metabolism and repair (eg Pol32, Pif1, Rad51).\nFollowing this study, the range of cellular functions that will be investigated is expected\nto expand greatly, thus representing an influential contribution to the field.\nBelow are relatively minor comments, curiosities, suggestions that the authors and\neditors might wish to consider in a revised manuscript.\nResponse: We thank Review #3 for the positive comments on our paper.\nReviewer 3, Comment 21. The authors did a genomic (CHEF) analysis (Fig. 5) of the\nLeu- Ade+ clones derived from the nup mutants and showed that some of them were\nnot regular BIR but instead contained some GCRs. That analysis was quite informative,\nthus raising the question: Was this type of genomic analysis also done for the bub\nmutants (Fig. 2,3,4)? If not, should it be done? Given that the SAC and SPOC pathways\nwere the major focus of the work following the screening phase, I think it might be\nimportant to add some information about this. In other words, how sure are the authors\nthat Leu- Ade+ clones derived mutants such as bub2D or bfa1D actually mostly carry\nBIR products?\nReviewer 3, Response 21. In response to this concern, we conducted CHEF analysis\nfor all SAC and SPOC mutants; specifically, for bub1Δ, bub3Δ, mad2Δ, bub1Δmad2Δ,\nrad9Δ, rad9Δmad2Δ, bfa1Δ, bub2Δ, bub2Δmad2Δ, nup188Δmad2Δ, nup188Δrad9Δ,\nnup188Δnup84Δ. For each of these strains, we analyzed ~20 Leu-Ade+ outcomes from\nYEP-Gal plates (268 in total). These new data were added to Supplementary Figures 4-\n6.\nReviewer 3, Comment 22. Another aspect that I think should be commented on is the\nremarkable absence of half-crossovers (HC) in bub2D and nup184D. Both of these\nmutants displayed a massive drop in viability following DSB induction, and in both HCs\nwere almost entirely gone. Is there a relationship? The authors included some\ndiscussion about gene conversions (GC), pointing out that GCs are in a “fast” repair\n13\nclass of products. HCs might also be in that “fast” repair group, so why are HCs gone\nwhile GCs increase (in bub2D)? I think this warrants some discussion.\nReviewer 3, Response 22. We thank the reviewer for this insight. As we mentioned in\nour response #10, we predicted that any delay or interruption of BIR should promote cell\ndeath in the absence of Bub2. The formation of HCs is likely associated with additional\ndelays during BIR, such as BIR pausing, interruption, resolution of BIR intermediates,\nand a secondary DSB (breakage of BIR donor), which could extend nuclear division\narrest. Therefore, we would expect that HC events would not complete before the onset\nof cytokinesis and would instead be associated with cell death. We believe that the\nabsence of HCs in nup188∆ mutants (we believe this is the gene the reviewer intended,\nversus NUP184) could be explained in a similar way, which is supported by our new\nobservation that mad2Δ and rad9Δ partially suppress cell lethality after DSB initiation in\ncells lacking Nup188 (Fig. 5a).\nIn response to this comment, we included the following sentence in our Results section:\n• “The fraction of HC outcomes among survivors was very low in bub2Δ and bfa1Δ\nmutants (Fig. 4d), likely because of the extended time required for HC formation,\nwhich involves secondary breakage of the donor chromosome.”\nReviewer 3, Comment 23. -Finally, and not as relevant, I was a bit intrigued by the\nmeaning of flower and bee in Figure 1e. I am not against it, but I did not find any\ncomment in the manuscript or the figure legend to explain its meaning. I suggest the\nauthors add some comment to provide context to the readers about why the diagram\nwas drawn as such.\nReviewer 3, Response 23. To eliminate any possible confusion, we replaced the flower\nwith a table for a more conventional presentation of the data. Please see the table in\nFigure 1e.\nReferences:\nCaydasi, A. K., B. Ibrahim and G. Pereira, 2010 Monitoring spindle orientation: Spindle\nposition checkpoint in charge. Cell Division 5: 28.\nDotiwala, F., J. C. Harrison, S. Jain, N. Sugawara and J. E. Haber, 2010 Mad2 prolongs DNA\ndamage checkpoint arrest caused by a double-strand break via a centromere-\ndependent mechanism. Curr Biol 20: 328-332.\nHamed, M., and W. Antonin, 2021 Dunking into the Lipid Bilayer: How Direct Membrane\nBinding of Nucleoporins Can Contribute to Nuclear Pore Complex Structure and\nAssembly. Cells 10: 3601.\nHoyt, M. A., L. Totis and B. T. Roberts, 1991 S. cerevisiae genes required for cell cycle arrest\nin response to loss of microtubule function. Cell 66: 507-517.\nJain, S., N. Sugawara, J. Lydeard, M. Vaze, N. Tanguy Le Gac et al., 2009 A recombination\nexecution checkpoint regulates the choice of homologous recombination pathway\nduring DNA double-strand break repair. Genes Dev 23: 291-303.\nmanner, independently of the kinetochore. PLoS Genet 4: e1000015.\nLi, R., and A. W. Murray, 1991 Feedback control of mitosis in budding yeast. Cell 66: 519-\n531.\nWeiss, E., and M. Winey, 1996 The Saccharomyces cerevisiae spindle pole body\nduplication gene MPS1 is part of a mitotic checkpoint. J Cell Biol 132: 111-123.\nZhou, Y. F., P. D. Waterman, M. Ashton, S. Caban-Penix, G. Memisoglu et al., 2024\nProlonged Cell Cycle Arrest in Response to DNA damage in Yeast Requires the\nMaintenance of DNA Damage Signaling and the Spindle Assembly Checkpoint, pp.\nReviewer #1 (Remarks to the Author):\nThe revision addresses most of my comments and includes additional sets of experimental data. The authors'\nresponses are thorough and articulated. Overall, the current version is vastly improved.\nResponse: We thank Reviewer #1 for these nice comments.\nReviewer #2 (Remarks to the Author):\nThe revised study effectively addresses my initial concerns, is significantly improved, and will be a valuable\naddition to the BIR literature and related fields. I congratulate the authors for conducting robust revisions. I\nstill recommend the following minor changes to the text to help improve accuracy and clarity in a few\ninstances.\nResponse: We thank Reviewer #2 for these nice comments.\nLine 32: consider replacing \"nuclear arrest\" with a less vague term that is less broad. For example, some non-\nspecialist readers may interpret this as a transcriptional arrest only, etc. Therefore, I recommend being more\nspecific/clear.\nResponse: We made the suggested change.\nLine 35: regarding \"...during BIR: Nup84 acts prior to BIR synthesis....\", I recommend changing this to\n\"...during BIR: Nup84 acts before DNA synthesis and Nup188 functions later to support repair completion\".\nThis is to avoid terms like \"BIR synthesis\", which is more specialized and would be less clear to non-\nspecialists. Also, this change would avoid using \"BIR\" so many times in a single sentence.\nResponse: We made the suggested change.\nLine 370: Replace \"...suggesting that SWR1...\" with \"tentatively suggesting that, at least within this\nexperimental system, SWR1 may....\". This is recommended because the literature appears to suggest that the\nimpact of SWR1/HTZ1 on BIR may be context-dependent, and future studies would likely be necessary to\nprovide deeper, context-specific insights.\nResponse: We introduced the suggested change.\nLines 392-393: Replace \"...making it, to our knowledge, the first demonstration of the role of a DDC-to-SAC\ntransition in successful completion of DSB repair.\" with \"...uncovering a crucial role for the DDC-to-SAC\ntransition in the completion of DSB repair.\" I recommend avoiding words such as \"first\", \"new\", \"first time\",\netc.\nResponse: We introduced the suggested change.\nReviewer #3 (Remarks to the Author):\nThe authors have adequately responded to my initial review comments. I have no further concerns.\nResponse: We thank Reviewer #3 for these comments."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revision addresses most of my comments and includes additional sets of experimental data. The authors' responses are thorough and articulated. Overall, the current version is vastly improved.", "reviewer_2": "The revised study effectively addresses my initial concerns, is significantly improved, and will be a valuable addition to the BIR literature and related fields. I congratulate the authors for conducting robust revisions. I still recommend the following minor changes to the text to help improve accuracy and clarity in a few instances.\nLine 32: consider replacing \"nuclear arrest\" with a less vague term that is less broad. For example, some non-specialist readers may interpret this as a transcriptional arrest only, etc. Therefore, I recommend being more specific/clear.\nLine 35: regarding \"...during BIR: Nup84 acts prior to BIR synthesis....\", I recommend changing this to \"...during BIR: Nup84 acts before DNA synthesis and Nup188 functions later to support repair completion\". This is to avoid terms like \"BIR synthesis\", which is more specialized and would be less clear to non-specialists. Also, this change would avoid using \"BIR\" so many times in a single sentence.\nLine 370: Replace \"...suggesting that SWR1...\" with \"tentatively suggesting that, at least within this experimental system, SWR1 may....\". This is recommended because the literature appears to suggest that the impact of SWR1/HTZ1 on BIR may be context-dependent, and future studies would likely be necessary to provide deeper, context-specific insights.\nLines 392-393: Replace \"...making it, to our knowledge, the first demonstration of the role of a DDC-to-SAC transition in successful completion of DSB repair.\" with \"...uncovering a crucial role for the DDC-to-SAC transition in the completion of DSB repair.\" I recommend avoiding words such as \"first\", \"new\", \"first time\", etc.", "reviewer_3": "The authors have adequately responded to my initial review comments. I have no further concerns."}, "author_response": "Response: We thank Reviewer #1 for these nice comments.\nResponse: We thank Reviewer #2 for these nice comments.\nResponse: We made the suggested change.\nResponse: We made the suggested change.\nResponse: We introduced the suggested change.\nResponse: We introduced the suggested change.\nResponse: We thank Reviewer #3 for these comments."}]

Decision letter