DNA double-stand breaks (DSBs) pose a threat to genome integrity and cell viability. If unrepaired, DSBs can lead to cell death, and if repaired improperly, DSBs can lead to loss of genetic information or chromosomal rearrangements associated with various pathologies such as neurodegeneration and cancer (Moynahan and Jasin, 2010; Rass et al., 2007). Cells have two primary pathways to repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). HR is the high-fidelity mode of DSB repair because it uses a homologous template, generally the sister chromatid, for repair. HR is essential for maintaining genome integrity. For example, there is increased sensitivity to DNA damaging agents and increased frequencies of chromosome rearrangements in HR-deficient yeast cells (Putnam and Kolodner, 2017; Symington et al., 2014). Furthermore, defects in human HR proteins, such as BRCA1 and BRCA2, are associated with increased risk for breast and ovarian cancers, as well as Fanconi anemia (Prakash et al., 2015; Taylor et al., 2019; Wang et al., 2015).

To repair DSBs by HR, the Mre11-Rad50-Xrs2/NBS1 (MRX/N) complex, stimulated by CDK-phosphorylated Sae2/CtIP, nicks the 5′-terminated strands on either side of the DSB, followed by 3′–5′ exonucleolytic processing back towards the break ends (Cannavo and Cejka, 2014; Garcia et al., 2011). This end-clipping reaction is followed by further processing from the 5′ ends by Exo1 or Dna2-Sgs1/BLM to create longer 3′ overhangs (Cejka and Symington, 2021). This second step, termed long-range resection, has been shown to be required for several HR processes, including single-strand annealing (SSA), yeast mating-type switching and interchromosomal gene conversion (Gobbini et al., 2020; Guo et al., 2017; Mimitou and Symington, 2008; Zhu et al., 2008). In Saccharomyces cerevisiae, resection initiation by MRX is essential to process DSBs with end-blocking lesions, whereas clean DSBs can be processed directly by the long-range resection machinery in the absence of MRX, albeit with delayed kinetics (Cejka and Symington, 2021). Once initiated, resection proceeds at about 4 kb/h in cells that lack a homologous template for repair (Chung et al., 2010; Zhu et al., 2008). Measurements of resection tract lengths in cells undergoing recombination have primarily utilized site-specific endonucleases, which cleave both sister chromatids, thus preventing use of the preferred donor duplex to template HR. By using a donor allele that lacks the endonuclease cleavage site, either in diploids or haploids with repeats on different chromosomes, average resection tracts of >2 kb in length were reported and resection tract length correlated with the time required for repair (Chung et al., 2010). Consistent with this finding, a more recent study found that resection tracts were short for a rapid sister chromatid repair event (Jakobsen et al., 2019).

Although long-range resection is part of canonical models of repair by HR, the physiological role of this process is not completely understood given that several lines of evidence suggest minimal homology or reduced resection is sufficient for HR. For example, spontaneous recombination can occur with ~250 bp of homology (Jinks-Robertson et al., 1993). Additionally, efficient gene conversion can occur with as little as 250 bp of homology on either side of a programmed DSB (Inbar et al., 2000), which is within the range of resection tract lengths produced by MRX-catalyzed short-range resection (Cannavo et al., 2019; Gnügge et al., 2023; Mimitou et al., 2017). Taken together, these results indicate that recombination can occur through much shorter tracts of ssDNA than are produced by long-range resection. Indeed, it has been shown that in G2-phase exo1∆ sgs1∆ cells, repair efficiency in response to ionizing radiation is only slightly reduced and kinetics are delayed by around one hour compared to wild-type (WT) (Westmoreland and Resnick, 2016). Furthermore, diploids lacking Exo1 or its nuclease activity exhibit near WT levels of joint molecule formation and meiotic divisions even though resection tract lengths are greatly reduced (~270 nucleotides in exo1-nd cells compared with ~800 nucleotides in WT; Mimitou et al., 2017; Zakharyevich et al., 2010). These findings suggest that long-range resection may not be necessary for HR in all scenarios; however, the reason for this context dependence remains unclear.

In addition to generating ssDNA substrates for Rad51-catalyzed HR, the end resection machinery is associated with DNA damage checkpoint signaling (Waterman et al., 2020). The MRX/N complex recruits and activates the Tel1/ATM kinase in response to DSBs, after which signaling transitions to Mec1/ATR once resection generates sufficient ssDNA for RPA and Ddc2/ATRIP binding (Shiotani and Zou, 2009; Waterman et al., 2020). Once activated, the DNA damage checkpoint limits extensive resection to prevent accumulation of excessive ssDNA through multiple mechanisms. Rad9/Crb2 antagonizes the Dna2-Sgs1 mechanism in yeast (Bonetti et al., 2015; Ferrari et al., 2015; Lazzaro et al., 2008; Leland et al., 2018), while Rad53, the effector kinase for Tel1 and Mec1, inhibits Exo1 activity by phosphorylation of the C-terminal regulatory domain (Morin et al., 2008; Yu et al., 2018). The 9-1-1 DNA damage clamp (Ddc1, Mec3, and Rad17 in budding yeast) and Rad24, the large subunit of the 9-1-1 clamp loader complex, attenuate resection by MRX and promote resection by Exo1 (Gobbini et al., 2020; Ngo et al., 2014; Ngo and Lydall, 2015). The Tel1 and Mec1 kinases positively influence resection initiation by MRX and Sae2 but limit extensive resection by promoting recruitment and retention of Rad9 to chromosome in the vicinity of DSBs (Cannavo et al., 2018; Cartagena-Lirola et al., 2006; Waterman et al., 2020).

The DNA damage checkpoint and end resection are also linked to chromosome mobility. DNA damage induces mobility of the broken chromosome (local), and, to a lesser extent, mobility of undamaged chromosomes (global; Dion et al., 2012; Miné-Hattab and Rothstein, 2012; Seeber et al., 2013). The checkpoint proteins Mec1 and Rad9 are necessary for local and global DNA damage-induced chromosome mobility (Dion et al., 2012; Seeber et al., 2013). Additionally, checkpoint activation in the absence of DNA damage is sufficient to increase chromosome mobility (Seeber et al., 2013). Chromosome mobility is also linked to end resection, as deletion of Sae2, which delays resection, also delays chromosome mobility (Miné-Hattab and Rothstein, 2012). Therefore, resection, checkpoint activation, and chromosome mobility likely collaborate to facilitate DNA repair, especially when a repair template is not in close proximity to the broken chromosome.

Given that the requirement for long-range resection seems to be context dependent, we wanted to explore further the role of long-range resection in HR. Here, we show that interchromosomal gene conversion is significantly impaired in the absence of long-range resection, consistent with previous findings (Gobbini et al., 2020; Guo et al., 2017). Remarkably though, we find that intrachromosomal recombination between closely linked repeated sequences occurs at almost WT levels when long-range resection is eliminated, providing evidence that long-range resection is dispensable under certain circumstances. Thus, the ssDNA tracts exposed by MRX-Sae2 alone must be sufficient to facilitate HR, and the requirement for long-range resection in the interchromosomal context must go beyond simply exposing adequate ssDNA. We suggest that the main role for long-range resection in mediating interchromosomal recombination is in DNA damage checkpoint activation. When the DSB and repair template are spatially separated, cells require time and a more active homology search facilitated by chromosome mobility, and therefore long-range resection is necessary. When the DSB and repair template are in close proximity, the homology search is rapid enough that the need for checkpoint activation and chromosome mobility is circumvented, and long-range resection is dispensable.

Current models of HR include MRX-Sae2 catalyzed resection initiation, followed by long-range resection by Exo1 and/or Dna2-Sgs1. Here, we show that long-range resection is dispensable for DSB-induced HR when a repair template is located in close proximity on the same chromosome, suggesting that sufficient ssDNA is generated by MRX-Sae2 for Rad51-catalyzed repair. Long-range resection becomes crucial for efficient recombination when the donor allele is located on a different chromosome or greater than ~50–100 kb from the DSB site on the same chromosome where the DNA damage checkpoint is activated (Figure 1, Figure 5). The necessity for long-range resection in these scenarios is consistent with previous work showing a requirement for Exo1 and Sgs1-Dna2 in interchromosomal repair (Gobbini et al., 2020; Guo et al., 2017). Our results provide a possible explanation for why others have observed long-range resection-independent recombination (Westmoreland and Resnick, 2016; Zakharyevich et al., 2010). First, when G2 cells are treated with IR, a broken chromatid is likely in close proximity to its sister, facilitating the homology search and subsequent repair. Second, multiple DSBs are induced by IR and during meiosis, and even the limited resection at each of these sites is likely to cumulatively yield sufficient ssDNA to activate the checkpoint (Gobbini et al., 2020).

One caveat to our findings and those of others reporting long-range resection independent recombination is that resection tracts produced by MRX under physiological conditions are likely to be shorter than those formed in exo1∆ sgs1∆ cells due to engagement of the long-range resection machinery (Mimitou and Symington, 2008; Zhu et al., 2008). Thus, long-range resection may be playing a more important role than is apparent in cells lacking Exo1 and Sgs1.

Given that the DNA damage checkpoint is activated in scenarios where repair is delayed, long-range resection may act as a timing mechanism for checkpoint activation. Relatively quick repair circumvents the need for the checkpoint. However, if repair takes longer, long-range resection serves to create sufficiently long ssDNA tracts for checkpoint activation. In the absence of long-range resection, and therefore robust checkpoint activation, cells may proceed through to the next cell cycle with a broken chromosome, leading to cell death (Figure 6). Artificial G2/M arrest of exo1∆ sgs1∆ cells is insufficient to rescue the HR defect, indicating that the DNA damage checkpoint is responsible for eliciting effects other than cell cycle delay in order to promote HR. A more active role for the DNA damage checkpoint is supported by our finding that artificial induction of the checkpoint can partially rescue the interchromosomal repair defect of exo1∆ sgs1∆ cells (Figure 4B). The rescue we see is likely incomplete due to the short galactose induction time used to avoid a dominant negative effect of the checkpoint system. Additionally, we do not know whether inducing the checkpoint elsewhere in the genome will have the same effect on mobility at the DSB site as the native checkpoint response. Finally, there is likely some effect of having short resection tracts that limits DSB mobility as well, possibly through inefficient Rad51 loading (Figure 3D, Figure 5—figure supplement 2).

Figure 6

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The more substantial recombination deficiencies of mec1∆ sml1∆, rad53∆ sml1∆, rad9∆, and rad24∆ cells in the interchromosomal versus the intrachromosomal context further supports the role of the checkpoint in the former, but not the latter scenario. As noted above, the failure of these mutants to facilitate efficient interchromosomal recombination is due to their checkpoint defect and not to a resection defect. The observation that loss of Rad9 or Rad24 in long-range defective cells is either epistatic or suppressive, respectively, supports the checkpoint defect in exo1∆ sgs1∆ cells. Restoring the checkpoint by expression of a Ddc2-Rad53 fusion protein in rad9∆ cells rescued the interchromosomal defect indicating that Rad9 is not required for HR per se and its function is to activate Rad53. It is also interesting to consider that the checkpoint, and therefore Mec1 and Rad9, are necessary for DNA damage-induced chromosome mobility (Dion et al., 2012; Seeber et al., 2013). Rad24 is linked to chromosome mobility since the loading of the 9-1-1 complex at resected DNA ends is necessary for Mec1 activation, and therefore checkpoint signaling (reviewed in: Finn et al., 2012). Given that interchromosomal recombination is likely to be more spatially challenging than proximal intrachromosomal repair, it is reasonable to think that the need for chromosome mobility would be increased for interchromosomal recombination. Consistent with this idea, we do observe a partial rescue of interchromosomal recombination in the absence of Exo1 and Sgs1 by overexpressing Rad51. This result likely reflects the fact that Rad51 is necessary for chromosome mobility and that this mobility helps facilitate repair (Dion et al., 2012; Kalocsay et al., 2009; Miné-Hattab et al., 2017; Miné-Hattab and Rothstein, 2012; Smith et al., 2018). Rad51 loading stiffens and extends ssDNA (Ogawa et al., 1993; Sung and Robberson, 1995), possibly reducing the complexity of the homology search as has been shown recently for RecA in bacteria (Wiktor et al., 2021). The rate limiting step of Rad51 filament formation is the nucleation step, which requires ~6 Rad51 monomers, and increased concentrations of Rad51 promote nucleation in vitro (Candelli et al., 2014; Miné et al., 2007; Paoletti et al., 2020; Qiu et al., 2013). The increased Rad51 concentration likely increases the probability of nucleation on short resection tracts, thereby promoting filament polymerization for homology search. Interestingly, it was recently shown that distal global mobility is facilitated by and enhances HR and is dependent on Rad51 and Rad9, while proximal global mobility occurs independent of these factors and is dispensable for HR (García Fernández et al., 2022). Given that long-range resection is an important upstream step of both checkpoint activation and Rad51 loading, this result may explain why we observe that long-range resection is required for distal (interchromosomal) recombination but is dispensable for proximal (intrachromosomal) recombination.

We find that WT cells repair the I-SceI break at high efficiency, independent of the donor location (Figure 5A, Figure 5—figure supplement 1). This is in contrast to findings that showed differences in both intra- and interchromosomal repair efficiency that correlated with contact frequency in WT cells (Agmon et al., 2013; Lee et al., 2016; Wang et al., 2017). Although the difference between these results and our own is unclear, it may be due to donor homology and/or use of HO versus I-SceI. Donor size positively impacts repair efficiency (Lee et al., 2016), and our system contains significantly more homology than prior studies (3.7 kb vs 2 kb or 1.2 kb) (Agmon et al., 2013; Lee et al., 2016). The difference in cutting efficiency of the endonuclease may also influence repair proficiency as determined by plating assays. Since I-SceI cleavage is less efficient than HO, some cells might divide on galactose-containing medium before the DSB is induced and survival of only one daughter cell would be required for colony formation leading to an over-estimation of repair efficiency. Based on our results, it seems that regardless of the donor position, sufficient end resection and checkpoint activation can facilitate repair, accounting for the fact that we only see differences in repair efficiency in the absence of long-range resection.

The failure to robustly rescue interchromosomal recombination deficiency of long-range resection deficient cells by using higher contact donors was unexpected at first, given previous work (Lee et al., 2016). However, even the higher contact frequency interchromosomal donors that were used are still relatively low compared to intrachromosomal contacts within Chr XV (Lazar-Stefanita et al., 2017). Hence, it might not be as surprising that we only observed improvement of recombination efficiency for the closest donor sites on the same chromosome. We also do not see an effect of centromere proximity on repair that was reported previously (Wang et al., 2017). However, this could be due to not using donors within 240 kb of the centromere. Nevertheless, our intrachromosomal data suggest that linear distance between the DSB site and donor site is the primary determinant of repair efficiency in the absence of long-range resection. Together, these results demonstrate that cells deficient for long-range resection are only proficient for recombination when the recombining sequences are in close proximity, but that WT cells can facilitate recombination independent of donor proximity.

Based on our results, it becomes clear that the importance of long-range resection does not necessarily lie in its ability to expose multiple kilobases of homology, although there may be certain contexts in which this is important. For example, long-range resection promotes the usage of more extensive stretches of homology, even if they are not in the immediate vicinity of the DSB. This may be especially important if a DSB occurs in a repetitive element, as long-range resection should suppress usage of short homologies between repeats and favor recombination with more extensive homologies in the surrounding sequence. Long-range resection may also promote a more efficient homology search by permitting invasion of multiple substrates (Wright and Heyer, 2014). However, this multi-invasion process has also been shown to be mutagenic, so there may be a trade-off (Piazza et al., 2017; Ruiz et al., 2009). Additionally, long-range resection has been shown to suppress telomere addition at slowly-repaired DSBs (Chung et al., 2010; Lydeard et al., 2010). Although we cannot exclude the possibility that loss of recombinants in the exo1∆ sgs1∆ interchromosomal assay is due to de novo telomere addition, it is unclear why this would not also occur in the intrachromosomal system.

We suggest that an additional function of long-range resection is to activate the checkpoint when repair is delayed, thereby increasing chromosome mobility and promoting the search for homology. This function may be relevant in a context in which recombination with the sister chromatid is inefficient, thus necessitating repair with a non-allelic donor, potentially resulting in a compromise between cell survival and genome integrity. Despite the circumvention of long-range resection in scenarios of rapid repair, this process clearly serves an important purpose in multiple contexts and is supported by the evolutionary maintenance of two redundant pathways to create longer tracts of ssDNA.

All data generated or analyzed during this study are included in the manuscript and supporting files; Source data files have been provided for Figures 1-5 and associated figure supplements.

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Author details

1. Michael T Kimble

   1. Program in Biological Sciences, Columbia University, New York, United States
   2. Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8042-2868

2. Matthew J Johnson

   1. Program in Biological Sciences, Columbia University, New York, United States
   2. Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4312-5618

3. Mattie R Nester

   Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, United States

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

4. Lorraine S Symington

   1. Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, United States
   2. Department of Genetics & Development, Columbia University Irving Medical Center, New York, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   lss5@cumc.columbia.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1519-4800

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