Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Apr 23.
Published in final edited form as: Mol Cell. 2010 Apr 23;38(2):211–222. doi: 10.1016/j.molcel.2010.02.028

Molecular structures of crossover and noncrossover intermediates during gap repair in yeast: implications for recombination mechanisms and the regulation of recombination fidelity

Katrina Mitchel 1,1, Hengshan Zhang 1,1, Caroline Welz-Voegele 1, Sue Jinks-Robertson 1,*
PMCID: PMC2865147  NIHMSID: NIHMS190924  PMID: 20417600

SUMMARY

The molecular structures of crossover (CO) and noncrossover (NCO) intermediates were determined by sequencing the products formed when a gapped plasmid was repaired using a diverged chromosomal template. Analyses were done in the absence of mismatch repair (MMR) to allow efficient detection of strand-transfer intermediates, and the results reveal striking differences in the extents and locations of heteroduplex DNA (hDNA) in NCO versus CO products. These data indicate that most NCOs are produced by synthesis-dependent strand annealing rather than by a canonical double-strand break repair pathway, and that resolution of Holliday junctions formed as part of the latter pathway is highly constrained to generate CO products. We suggest a model in which the length of hDNA formed by the initiating strand invasion event determines susceptibility of the resulting intermediate to antirecombination and ultimately whether a CO- or a NCO-producing pathway is followed.

INTRODUCTION

In mitotically-dividing cells, homologous recombination is important for the repair of double-strand breaks (DSBs) generated directly by agents such as ionizing radiation, as well those that arise spontaneously through encounters of the replication machinery with DNA damage. In meiosis, the repair of programmed DSBs via recombination between homologous chromosomes is essential for proper chromosome segregation. The basic mechanisms of recombination are highly conserved between mitosis and meiosis, and the ability to study both has made the yeast Saccharomyces cerevisiae a particularly attractive model system. Here, we use the repair of a gapped plasmid in mitosis to model DSB repair in yeast.

Multiple mechanisms of homologous recombination are used to repair DSBs in yeast (for reviews see Paques and Haber, 1999; San Filippo et al., 2008; Symington, 2002), and all begin with resection of the 5′ ends to yield 3′ single-stranded tails. In the classic DSB repair (DSBR) model of recombination (Szostak et al., 1983), one of the 3′ tails thus exposed invades a homologous duplex DNA molecule, creating a region of heteroduplex DNA (hDNA) and a displaced, single-stranded D-loop (Figure 1, step A). Extension of the invading 3′ end by DNA polymerase enlarges the D-loop, thereby exposing sequences complementary to the other side of the original DSB. Annealing of the D-loop to the other (noninvading) 3′ end yields an intermediate with two Holliday junctions (HJs; Figure 1, step B), the resolution of which either maintains the original linkages of sequences that flank the junctions (a noncrossover or NCO event) or switches the flanking sequences (a crossover or CO event). Resolution of HJs can occur by sequential nicking and ligation reactions, or can be mediated by the combined action of a helicase and topoisomerase (Figure 1, steps C and D, respectively). Whereas the former mechanism is generally assumed to generate both CO and NCO products, the latter “dissolution” mechanism generates only NCOs. In the synthesis-dependent strand annealing (SDSA) model, the D-loop collapses and the extended 3′ end is displaced from the invaded duplex, allowing it to anneal to the single-stranded tail on the other side of the DSB (Figure 1, step E). Because HJs are not formed, SDSA results only in NCO events. A distinguishing feature of DSBR is the formation of hDNA on both sides of the repaired break; in SDSA only a single tract of hDNA persists in the repaired products (gray boxes in Figure 1).

Figure 1.

Figure 1

Open in a new tab

SDSA and DSBR models of gap repair. Solid blue and orange lines correspond to single strands of the gapped and intact alleles, respectively; arrowheads represent the 3′ ends of DNA strands. Dotted lines correspond to new DNA, which is colored according to the template directing its synthesis. The solid triangle at the distal end of the D-loop formed in step A represents the position of Rad1-Rad10 cleavage to facilitate second-end capture and generate an intermediate with a single HJ. With the double HJ, a CO results when the exchanged strands are nicked at one junction and the unexchanged strands are nicked at the other junction; the CO position occurs at the junction where the unexchanged strands are nicked. Gray boxes indicate the positions of hDNA in intermediates and products.

Recombination is a homology-driven process that can involve sister chromatids, homologous chromosomes or ectopic sequences dispersed throughout the genome. While interactions between identical sister chromatids are typically of no genetic consequence, recombination between homologs can uncover recessive markers and ectopic interactions can generate a wide variety of genome rearrangements. When mitotic recombination occurs, it clearly is important that it involve the “correct” sequences in order to avoid undesirable genetic outcomes. The rate of ectopic recombination in yeast is directly proportional both to the length of total homology (Jinks-Robertson et al., 1993) and to the degree of sequence identity (Datta et al., 1997) between the substrates.

When interacting sequences are not identical, an hDNA intermediate can contain mismatches that are processed by the mismatch repair (MMR) machinery. The repair of such mismatches results in the classic genetic phenomenon of gene conversion, which is defined as the unidirectional transfer of information from one DNA duplex to another. If mismatches fail to be repaired, the mismatched strands will segregate at the next round of replication, resulting in a sectored colony. In addition to initiating a simple repair process, mismatches in hDNA intermediates can block the production of mature recombinants (reviewed by Surtees et al., 2004). The enforcement of stringent identity requirements during recombination derives primarily from antirecombination activity of the MMR system, a system best characterized with respect to its role in correcting DNA synthesis errors. In yeast, the major players in nuclear mismatch recognition/repair are Msh2, Msh3 and Msh6, which are MutS homologs, and the MutL homologs, Pms1 and Mlh1 (for reviews see Harfe and Jinks-Robertson, 2000; Kunkel and Erie, 2005). In addition to these core MMR proteins, the repair of replication errors involves a 5′ > 3′ exonuclease (Exo1) and the PCNA sliding clamp. Whereas the core MMR proteins and Exo1 are also important for regulating mitotic recombination fidelity in yeast (e.g., see Nicholson et al., 2000), PCNA appears to play little, if any, role in this process (Stone et al., 2008). In addition, the Sgs1 helicase is important in antirecombination (Myung et al., 2001; Spell and Jinks-Robertson, 2004; Sugawara et al., 2004), but has no known function during the general repair of mismatches. These genetic studies suggest a basic mechanistic difference between the replication- and recombination-related activities of the MMR system.

In the current study, gapped plasmids were transformed into a haploid, MMR-defective strain containing a diverged chromosomal repair template. Following the selection of recombinants, the extent of hDNA in CO and NCO intermediates was inferred by sequencing the products derived from individual repair events. Results demonstrate that the structures of CO and NCO intermediates are distinctly different, an observation most easily explained if COs and NCOs are generated by DSBR and SDSA, respectively. Additional examination of gap-repair products generated in the presence of MMR provides molecular confirmation that the MMR-directed correction of mismatches in hDNA is distinct from MMR-associated antirecombination.

RESULTS

The transformation-based gap-repair system used previously to examine the effect of sequence divergence on mitotic CO and NCO events is shown in Figure 2 (Welz-Voegele and Jinks-Robertson, 2008). In this system, a plasmid-encoded HIS3 gene contains a gap that must be repaired using a 2%-diverged chromosomal template (a his3Δ3′ allele integrated at the CAN1 locus on chromosome V; see Figure S1 for sequence polymorphisms) in order to generate a His+ transformant. Following the selection of His+ colonies, CO and NCO events are distinguished based on the relative stability and instability, respectively, of the plasmid-encoded URA3 marker. To preserve hDNA in NCO versus CO intermediates, recombination products were initially derived in an MMR-deficient (mlh1Δ) background. In the absence of mismatch correction, hDNA is resolved by DNA replication to yield a sectored colony in which half of the cells contain one allele and half contain the other allele. To allow the detection of both alleles, DNA was isolated from His+ transformants without prior colony purification. In the case of NCO recombinants, both the reconstituted, plasmid-encoded HIS3 allele and the chromosomal repair template (recipient and donor alleles, respectively) were sequenced; in CO recombinants, both chromosomal alleles produced by plasmid integration were sequenced. Finally, to examine the antirecombination-related function of the MMR system, recombinants also were derived in an MMR-competent host strain.

Figure 2.

Figure 2

Open in a new tab

Gap-repair system. The plasmid contains an ARS, the URA3 gene (gray) and a gapped his3 allele (orange). The his3Δ3′ allele is (blue) located at the CAN1 locus on chromosome V. NCO and CO events generate His+ transformants with an unstable and stable Ura+ phenotype, respectively. The positions of primers used to selectively amplify recombination products are indicated.

NCO intermediates in the BglII gap-repair system

In the BglII system used previously, removal of BglII fragment from the HIS3 coding sequence generates an approximately 60-bp gap relative to the chromosomal repair template (see Figure S1). This gap is asymmetrically positioned within the region of donor homology, with 600 and 160 bp of homology upstream and downstream of the gap, respectively. In addition, the first upstream polymorphism is farther away from the gap than the first downstream polymorphism (approximately 80 and 30 bp, respectively). In the 91 NCO products analyzed, all sequence transfer was confined to the repaired allele; we did not detect any acquisition of plasmid sequences by the chromosomal donor allele. The structures of the repaired alleles are presented in Figure 3A. The presence of two complementary donor strands in the recipient allele corresponds to a gene conversion event, the extent of which is indicated by a blue donor segment in an otherwise yellow recipient allele. Sequence flanking the gap should be exchanged in the form of single strands to yield hDNA intermediates, and tracts are indicated as green segments. There was transfer of donor sequences flanking the gap in 75% (68/91) of the repaired plasmids and hDNA was detected >95% of the time (66/68). The few examples of gene conversion of markers flanking the gap could reflect nucleolytic expansion of the original gap, a failure to detect both products following replicative hDNA resolution, or a repair system that acts in the absence of the canonical MMR system.

Figure 3.

Figure 3

Open in a new tab

NCO products generated in the absence of MMR. The chromosomal his3Δ3′ and gapped alleles are indicated at the top of each panel in blue and yellow, respectively. Gap positions are indicated by the long, vertical blue lines; the positions of silent sequence polymorphisms are indicated by the short, vertical black lines in the gapped alleles (see Figure S1 for sequence changes). Each horizontal line corresponds to an independent gap-repair event, and the extent of DNA transferred from the chromosome is indicated. Green lines correspond to hDNA and blue lines to gene conversion events. Tract endpoints were placed between the most gap-distal polymorphism transferred and the next, unchanged polymorphism.

Of the 66 plasmids with hDNA, ~90% acquired donor sequence on only one side of the gap. The strong bias for unidirectional hDNA suggests that most NCO events were derived from SDSA rather than from DSBR, and hence did not involve an HJ-containing intermediate. Although an alternative interpretation is that the accompanying hDNA on the other side of the gap is usually too short to be detected, additional results with the BssHII system indicate that bidirectional hDNA is indeed rare (see below). In the SDSA model, the hDNA formed by invasion of the donor is dismantled and the newly-extended 3′ end anneals to the single-stranded tail on the other side of the DSB. The single tract of hDNA is thus on the side of the break opposite the side that initiates strand invasion (Figure 1), and the extent of hDNA in the product reflects the amount of DNA synthesis primed from the invading end. All but one of the unidirectional hDNA tracts was downstream of the gap, indicating that most of the initiating strand invasion occurred upstream of the gap. We note that the upstream side has the most total homology as well as the greatest length of gap-adjacent uninterrupted sequence identity, either of which could potentially bias the distribution of unidirectional hDNA.

When SDSA is initiated by strand invasion upstream of the gap, the invading 3′ end must be extended 60 nt to traverse the gap, plus an additional distance to allow annealing to the single-stranded tail on the other side. In any given NCO event, DNA synthesis extends past the most gap-distal polymorphism transferred, but presumably not as far as the next polymorphism. The average length of DNA synthesis associated with strand invasion was calculated by assuming that synthesis began precisely at the upstream edge of the gap and end halfway between the two relevant polymorpohisms. We estimate that the invading end was used to prime ~170 nt of DNA synthesis from the donor template. Although we assume here for simplicity that hDNA length directly reflects the extent of DNA synthesis from the invading end, is also possible that it instead reflects the extent of end resection on the annealing side of the gap. In the latter case, the invading end could be over-replicated relative to the end to which it anneals, which would require removal of the over-replicated end before ligation.

Although most NCOs in the BglII system had an hDNA tract, there were 23 events in which no hDNA was detected. If these reflect a failure to detect segregated hDNA, then there should be an equivalent number of NCOs with only donor polymorphisms (i.e., gene conversions). Only two gene conversions were detected, however, suggesting that most of the “no hDNA” class reflects short tracts of DNA synthesis that did not extend to the first polymorphism. In these cases, it is impossible to discern whether the initiating strand invasion occurred upstream or downstream of the gap. Finally, there were four NCOs with hDNA on both sides of the repaired gap, characteristic of a DSBR intermediate resolved by dissolution rather than by cleavage (Figure 1D).

NCO intermediates in the BssHII system

As noted above, the asymmetry of the gap in the BglII system could limit the length and/or direction of hDNA detected among NCOs. The presence of a 60-bp gap rather than a simple DSB could also affect recombination pathway choice and/or outcome. To address these issues, we used the same diverged his3 sequences to develope a second gap-repair system. This was accomplished by introducing a BssHII site near the center of the 820-bp donor-recipient homology (see Figure S1). Digestion of the resulting plasmid with BssHII prior to transformation generates an 8-bp gap relative to the chromosomal donor allele. In addition to the smaller gap, the first flanking polymorphisms are closer to the gap and are more symmetrically positioned than in the BglII system; both are ~20 bp from the BssHII-generated gap. As with the BglII system, no transfer of DNA from the broken plasmid to the chromosomal donor was detected among the 88 NCOs analyzed. The structures of the repaired plasmid alleles are shown in Figure 3B.

The locations of the hDNA tracts with respect to the gap were similar to those in the BglII system: 92% of the tracts detected were unidirectional (66/72), consistent with the SDSA mechanism, and there was a strong bias for the unidirectional tracts to be downstream of the gap (54/66). The small number of bidirectional tracts detected in the BssHII system (6/72), which are consistent with HJ dissolution rather than cleavage, was also similar to that observed with the BglII system (4/66). Eight of the unidirectional hDNA tracts were associated with contiguous, gap-proximal gene conversion tracts. Because of their hDNA association, these gene conversion tracts most likely reflect gap expansions. Using the downstream, unidirectional hDNA tracts to calculate DNA synthesis, we estimate an average length of ~235 nt of DNA synthesis in the BssHII system, which is ~40% longer than that in the BglII system. Finally, there were were seven unidirectional gene conversion tracts that were not associated with contiguous hDNA. The occurrence of an equivalent number of NCOs with no detectable hDNA suggests that these gene conversions likely reflect a failure to detect both of the sectors derived from hDNA segregation.

In the BssHII system, there were more unidirectional hDNA tracts that extended upstream of the gap than in the BglII system (12/72 versus 1/60, respectively). Although this could reflect a true shift to more upstream tracts in the BssHII system, a consideration of the average length of upstream tracts suggests an inability to detect comparable tracts in the BglII system. Only ~150 nt of DNA synthesis were primed when the invading end was downstream of the BssHII-generated gap; approximately 140 nt of DNA synthesis would be required to reach the first upstream polymorphism in the BglII system. We speculate that the abundant “no hDNA” class in the BglII system includes events initiated by downstream invasion, followed by limited DNA synthesis that failed to extend past the first polymorphism upstream of the gap.

CO intermediates in the BglII system

The extents of DNA transfer associated with CO events were determined by sequencing the full-length and truncated alleles generated by plasmid integration in the mlh1Δ background (Figure 4A). Strand transfer was detected in all 60 CO recombinants analyzed, and these can be divided into four classes. The most abundant class (34/60) was that in which hDNA was continuous on one side of the gap in one allele, and continuous on the other side of the gap in the other allele. This is precisely the pattern of hDNA predicted by the DSBR model (Figure 1, step C). Strikingly, hDNA was always present downstream of the gap in the full-length allele and always upstream of the gap in the truncated allele. As illustrated in Figure 5A, this pattern is expected only if the initiating strand invasion and the site of crossing over occur on the same side of the gap. The second class of CO events (9/60) contained continuous hDNA on only one side of the gap, and this single tract of hDNA adhered to exactly the same pattern as in the class 1 events: always upstream or downstream of the gap if present in the truncated or full-length allele, respectively.

Figure 4.

Figure 4

Open in a new tab

CO products generated in the absence of MMR. The cartoon above the sequenced events illustrates the products expected if the gap is repaired but no additional flanking sequence is transferred. Plasmid sequence is in yellow, chromosomal sequence in blue and hDNA in green. Classes 1–4 are described in the text.

Figure 5.

Figure 5

Open in a new tab

Relationship between the end that invades the donor, the CO position relative to the gap, and the location of hDNA in CO products. Dotted lines represent DNA synthesized during gap repair and are colored according to the template. Arrowheads represent the 3′ ends of DNA strands and gray boxes highlight the positions of hDNA. In Panel A, the donor allele is invaded by the 3′ end upstream of the gap, and intermediates with one or two HJs are shown. The single HJ postulated to result from D-loop nicking should always be upstream of the gap, as shown. Panel B illustrates nick-directed cleavage of single or double HJs. Filled circles correspond to 5′ ends at nicked HJs and the dashed black arrows indicate the positions where a nicked HJ is predicted to be cleaved by Mus81-Mms4. Such cleavage generates exclusively CO products. See Figure S2 for gene conversion tracts predicted if segregated hDNA fails to be detected.

In the DSBR model, one tract of hDNA reflects the initiating strand invasion event and the other is formed by second-end capture. Although is not possible to distinguish which hDNA tract corresponds to which event the first class of CO events can nevertheless be used to calculate the average length of hDNA upstream and downstream of the gap in intermediates. There are ~600 bp of total homology upstream of the gap, and the average upstream hDNA length, including DNA synthesis across the 60 bp gap, was ~520 bp. Downstream of the gap, where homology was limited to 160 bp, the average length of DNA synthesis was only ~180 bp.

A third class of CO events (9/60) had a gene conversion tract on only one side of the gap. We suggest that these events reflect only one-half of the sectored colony expected to form when a hDNA intermediate is resolved by DNA replication (see Figure S2). We again note that when a single conversion tract was present, it was always upstream or downstream of the gap in the truncated or full-length allele, respectively. Finally, there was a fourth class of CO events (8/60) that contained more complex events. Some of these had hDNA at the same positions in both alleles (“symmetric” hDNA), which can be explained by branch migration of an HJ. Others had a mix of hDNA and gene conversion tracts, and could result from MMR-independent repair. With regard to this possibility, an MMR-independent, short-patch repair pathway that corrects mismatches in recombination intermediates has been reported in S. cerevisiae (Coic et al., 2000) as well as in Schizosaccharomyces pombe (Kunz and Fleck, 2001).

CO intermediates in the BssHII system

Forty full-length and truncated alleles created by integration of the BssHII plasmid in an MMR-defective background are shown in Figure 4B. Most had either a tract of hDNA adjacent to the gap in each allele (class 1) or a single gene conversion tract (class 3). No COs with hDNA in only one allele (class 2 in the BglII system) were observed, perhaps because fewer events were examined and/or strand transfer was more efficiently detected with polymorphisms closer to the gap. Finally, there was a small number of class 4 events that had a complex pattern of hDNA and/or gene conversion tracts. Strikingly, the same pattern of strand transfer seen in the BglII system was present in the class 1 and 3 products: hDNA/gene conversion upstream and downstream of the gap in the truncated and full-length alleles, respectively. Using the class 1 events to calculate the average amount of DNA synthesis associated with CO intermediates, we estimate ~280 nt upstream and ~350 nt downstream of the BssHII-generated gap. The length of upstream DNA synthesis was less and the length of downstream synthesis greater in the BssHII system than in the BglII system (~520 nt and 180 nt, respectively), which likely reflects the relative positions of the gaps in the two systems. Despite the differences in gap positions and sizes, however, the overall patterns of strand transfer among COs in the BglII and BssHII system were remarkably similar.

Effect of MMR on gap repair in the BglII system

In the BglII system, most MMR-associated antirecombination activity is directed toward CO intermediates (Welz-Voegele and Jinks-Robertson, 2008; see also Tay et al., 2010). To provide insight into why CO intermediates are preferentially targeted and how antirecombination is effected, 59 CO and 90 NCO products derived from the WT background were sequenced (Figure 6). As in the mlh1Δ background, all sequence transfer in NCOs was from the chromosomal repair template to the gapped plasmid. In contrast to the persistent hDNA in the mlh1Δ background, however, only four examples of unrepaired hDNA were found among the 74 NCO recombinants with detectable strand transfer. Based on these data, we estimate that the efficiency of mismatch correction in NCO intermediates is greater than 90%. Although the persistence of hDNA was rare, it was present on both sides of the gap in three of the four bidirectional tracts, which are diagnostic of HJ dissolution. Because MMR was efficient among CO products derived from HJ cleavage (see below), an interesting possibility is that the repair of mismatches in DSBR intermediates is linked to junction cleavage.

Figure 6.

Figure 6

Open in a new tab

NCO and CO products of the BglII system in the presence of MMR. See legends to Figures 3 and 5 for NCO and CO details, respectively.

The distributions of gene conversion tracts in NCOs isolated in the presence of MMR (mostly unidirectional tracts extending downstream of the repaired gap) were very similar to those of hDNA tracts in the mlh1Δ background. This suggests that, as in the mlh1Δ background, SDSA is the primary mechanism for generating NCOs and that most of the initiating strand invasion occurs upstream of the gap in WT. Using the unidirectional gene conversion tracts downstream of the gap, we calculate that ~160 nt of DNA synthesis occurs in the WT background, an amount very similar to that estimated in the mlh1Δ background. Finally, the similar proportion of the no-transfer class in the WT and mlh1Δ backgrounds (29% and 25%, respectively) suggests that hDNA repair in NCO intermediates is strongly biased to favor the donated (chromosomal) allele. Altogether, the molecular comparison of NCO products derived in the presence versus absence of MMR indicates that mismatches affect neither the stability of the corresponding intermediates nor the mechanism that generates them.

The general patterns of strand transfer relative to the recombination-initiating gap also appeared similar among CO products isolated in the presence versus absence of MMR (compare Figures 4A and 6B). As in the mlh1Δ background, detectable strand transfer in the full-length and truncated CO products always occurred downstream and upstream of the gap, respectively, in the WT background. The average length of DNA synthesized upstream and downstream of the gap in the WT CO products was ~470 nt and ~180 nt, respectively, which is similar to that calculated in the mlh1Δ background (520 nt and 180 nt, respectively). Although hDNA was repaired in most NCOs isolated in the WT background, pure hDNA tracts persisted in ~22% (13/59) of the CO products. There were 12 tracts of hDNA upstream and 4 tracts downstream of the gap; unrepaired hDNA was present in both alleles in only three CO events. The repair efficiency of downstream hDNA tracts appears similar among CO and NCO products; the rarity of strand transfer upstream of the gap among NCO products precluded an estimation of repair efficiency. These data indicate that the MMR-dependent repair of hDNA occurs efficiently in those CO intermediates that escape MMR-dependent antirecombination activity, suggesting that the two processes are temporally distinct.

DISCUSSION

An implicit prediction of the DSBR model is the formation of hDNA on both sides of the initiating break, with each tract of hDNA being bordered by an HJ (Figure 1). The resulting double HJs can be cleaved to produce a NCO or CO outcome (step C), generating products which each contain hDNA (trans configuration). Since its inception, there have been several modifications to the DSBR model. First, it has been proposed that HJs can be resolved by helicase-driven dissolution, a process that generates only NCO products. In contrast to the trans hDNA associated with HJ cleavage, dissolution specifically predicts the presence of both hDNA tracts on the molecule with the initiating break (the cis configuration; step D in Figure 1). Second, the idea that NCO and CO events correspond to alternative modes of HJ cleavage has been challenged by the discovery that NCO can precede CO formation in both mitosis (Ira et al., 2003) and meiosis (Allers and Lichten, 2001). Consistent with the observed temporal separation, the SDSA mechanism of DSB repair provides a way to generate NCO events without going through an obligatory HJ intermediate (step E in Figure 1; Ferguson and Holloman, 1996; Nassif et al., 1994; Paques et al., 1998). Of particular relevance to the results reported here, the NCO-specific SDSA model predicts that hDNA will be present in only the repaired allele and that it will be limited to only one side of the gap.

In the present study, a diverged chromosomal allele was used as a template to repair a plasmid with a BglII- or BssHII-generated gap. Transformation experiments were done in the absence of MMR, thereby allowing the positions and extents of strand transfer in NCO and CO intermediates to be directly inferred by sequencing both products of individual recombination events. As discussed below, results suggest that NCOs result primarily from SDSA and that HJ cleavage is highly constrained to generate only CO products. In addition, repair products isolated in a WT background were examined for the BglII plasmid, for which the antirecombination activity of the MMR machinery has been shown to inhibit primarily CO events (Welz-Voegele and Jinks-Robertson, 2008). Results provide additional support for a model in which MMR-directed antirecombination is distinct from mismatch correction.

NCO events are produced primarily by SDSA

Despite differences in the initiating-gap size and position, the overall patterns of strand transfer in the BglII and BssHII systems were very similar. In the absence of MMR, hDNA was detected in the majority of the recipient, plasmid alleles, but no examples of hDNA were found in the corresponding chromosomal, donor alleles. A similar donor-to-recipient pattern of transfer has been observed during the repair of HO-induced breaks introduced into tandem repeats (Paques et al., 1998) and during mating type switching (Ira et al., 2006). The confinement of hDNA to the recipient allele in earlier studies was consistent with either the dismantling of D-loops or with HJ dissolution (steps D and E, respectively, in Figure 1), but these alternatives could not be distinguished. While the association of unidirectional gene conversion tracts with the mitotic repair of an HO-induced DSB (Palmer et al., 2003) and with meiotic recombination (Gilbertson and Stahl, 1996; Jessop et al., 2005; Merker et al., 2003) are consistent with D-loop collapse, studies done in the presence of MMR make it impossible to tell whether such conversion tracts reflect unidirectional hDNA or the manner in which bidirectional hDNA is repaired. A unique feature of our system is that it allows the location of hDNA relative to the initiating DSB in individual recombinants to be accurately assessed. More than 90% of the hDNA detected in NCO recombinants derived using either the BglII or BssHII gap-repair system was unidirectional, providing direct evidence that most NCOs are generated by SDSA rather than by HJ dissolution. Finally, it should be noted that the few NCO intermediates that did contain bidirectional tracts had the cis rather than the trans configuration of hDNA. The exclusive cis configuration suggests that HJ cleavage rarely generates NCOs in this system, which is consistent with the biased resolution of HJ-containing intermediates inferred in meiotic analyses (Allers and Lichten, 2001).

In the BglII system, there was a very striking asymmetry in the location of hDNA among NCO products, with >95% of the tracts extending downstream of the gap in the mlh1Δ strain (Figure 3). Because hDNA reflects second-end annealing during SDSA, invasion of the donor thus appeared to be almost exclusively initiated by the end located upstream of the gap (see step E in Figure 1). Additional data obtained with the BssHII system suggest that much of the perceived bias in the BglII system likely resulted from an inability to efficiently detect short, unidirectional upstream hDNA tracts. We note, however, that there was still a strong bias for unidirectional hDNA to extend downstream of the BssHII-generated gap and that a similar downstream bias has been reported during the repair of a chromosomal HO break (Palmer et al., 2003). Finally, the presence of shorter downstream hDNA tracts in the BglII than in the BssHII system indicate that asymmetry of the BglII-generated gap may have limited the extent of DNA synthesis following strand invasion.

CO events are produced by constrained cleavage of Holliday junctions

Random cleavage of double HJs is predicted to yield equivalent numbers of CO and NCO products, with the locations of the associated trans hDNA being random. Not only did we fail to detect a trans pattern of hDNA among ~200 NCO products sequenced, the hDNA in the full-length CO products was always located downstream of the gap and that in the truncated products was always upstream of the gap. This particular pattern of hDNA is predicted only if the CO site is on the same side of the gap as the single-stranded tail that initiates donor invasion (Figure 5A).

Both an exclusive production of CO products by HJ cleavage and the biased location of hDNA within these products can be explained if the resolution of HJ intermediates is nick-directed. Nicked HJs, which are precursors to mature, fully ligated HJs, are the preferred substrate for Mus81-Mms4 (Mus81-Eme1 in S. pombe and vertebrates) in vitro, and this complex is required for meiotic CO formation in S. pombe (Boddy et al., 2001). Importantly, Mus81-Mms4 uses the 5′ end of a nick near an HJ intermediate to direct cleavage of the opposing strand (Osman et al., 2003). As shown in Figure 5B, the net result of such directed cleavage is an obligatory CO that occurs on the side of the break that initiates strand invasion – exactly the pattern observed in our system. We thus speculate that Mus81-Mms4 may be responsible for resolving nicked HJs in this system, and predict that the positions of trans hDNA in CO products will be randomized upon its loss. Given the redundancy of HJ-resolving activities in budding yeast (Klein and Symington, 2009), however, it seems unlikely that Mus81-Mms4 will be absolutely required for CO production in our system.

CO events in the BglII system, as well as in similar gap-repair systems, are strongly dependent on the Rad1-Rad10 endonuclease (Schiestl and Prakash, 1988; Symington et al., 2000; Welz-Voegele and Jinks-Robertson, 2008), but this complex is not required for CO formation in other types of assays in budding yeast (Schiestl and Prakash, 1988). Rad1-Rad10 cleaves at junctions between single-stranded and duplex DNA in vitro (Bardwell et al., 1994), and it has been suggested that it might nick D-loops to facilitate second-end capture (Symington et al., 2000). Such an activity would generate an intermediate with a single HJ adjacent to the invading rather than the captured end (Figure 5), and hence at the position where the CO event presumably occurs in our system. An additional activity (e.g., Mus81-Mms4), however, would still be required to produce an exclusive CO outcome. An alternative possibility to D-loop processing is that Rad1-Rad10 directly processes HJs or that it removes the 3′ tails produced when the invading end is extended past the region of donor-recipient homology. With regard to a possible role of Rad1-Rad10 in processing recombination intermediates, it should be noted that single HJs are characteristic of meiotic recombination in S. pombe (Cromie et al., 2006) and that the Rad1 homolog (MEI-9) is required for most meiotic COs in Drosophila (Sekelsky et al., 1995).

MMR-directed antirecombination in the BglII system

In the BglII gap-repair system, antirecombination activity of the MMR system has a profound effect on the production of CO products, but little effect on NCOs (Welz-Voegele and Jinks-Robertson, 2008). The sequencing data reported here not only suggest that CO and NCO products are produced by distinct mechanisms, they also indicate that hDNA is more extensive in CO than in NCO intermediates. Although the associated mismatches could interfere with a late step that is unique to the DSBR pathway (e.g., HJ resolution or second-end capture by the displaced D-loop), we favor a model in which the initiating strand invasion event, which is common to the DSBR and SDSA pathways, is targeted by antirecombination. We suggest that the initiation of DSBR generally requires more extensive hDNA formation, and hence that early CO intermediates will be more efficiently detected and removed by the MMR machinery than early NCO intermediates. In terms of antirecombination mechanism, it is important to note that the rare CO events that escaped antirecombination in a WT background contained gene conversion rather than persistent hDNA tracts. This suggests that the mechanism of antirecombination is distinct from a canonical mismatch correction process. Interestingly, the MMR-PCNA interactions that are important in the removal of both replication- and recombination-generated mismatches appear to play little if any role during antirecombination (Stone et al., 2008). An attractive hypothesis is that the differing PCNA requirements reflect a temporal separation between antirecombination and recombination-associated mismatch correction, with PCNA-independent antirecombination occurring prior to the initiation of 3′-end extension, which depends on PCNA in vitro (Li et al., 2009).

A unifying model for mitotic gap repair

The data presented here bring together in a single assay observations made using a variety of systems, and these data form the basis of the model presented in Figure 7. The central feature of this model is that the length of hDNA formed upon invasion of the donor molecule is related to whether the DSBR or SDSA pathway will be followed, and hence whether a CO or NCO event will ultimately be produced. Long tracts of initiating hDNA would generally lead to DSBR and CO formation, while short hDNA would likely result in SDSA and a NCO outcome. We note that one version of SDSA invokes D-loop migration towards the extending end, which can collapse the D-loop and free the end to pair with a complementary 3′ tail on the other side of the DSB (Ferguson and Holloman, 1996). The initial D-loop size is expected to reflect the length of initiating hDNA, and we suggest that this size determines D-loop stability. Short hDNA tracts would be associated with a small D-loop that has a high probability of being dismantled by D-loop migration, thus favoring NCO production via SDSA. Longer hDNA, on the other hand, would generate a correspondingly larger D-loop that would persist longer and ultimately favor HJ formation and CO production. A specific model presented in Figure 7 is that length of hDNA formed upon invasion of the donor duplex directly reflects the length of the invading, single-stranded tail, which in turn will be determined by the extent of broken-end resection. The exonucleases responsible for end processing in yeast have recently been identified (Mimitou and Symington, 2008; Zhu et al., 2008), and it will be interesting to see if and how their presence/absence affects the NCO-CO outcome. We have shown that both Sgs1 and Exo1 are important in MMR-associated antirecombination and have assumed that this reflects a role in processing recombination intermediates (Nicholson et al., 2000; Spell and Jinks-Robertson, 2004). An alternative possibility is that the antirecombination activity derives from an end-resection role that promotes more extensive hDNA formation and increases the probability of mismatch detection.

Figure 7.

Figure 7

Open in a new tab

Model relating end resection to SDSA-DSBR pathway choice and MMR-directed antirecombination. Dotted lines represent new DNA, which is color-coded to reflect the template; gray donuts near the extended 3′ ends represent PCNA molecules. Opposed orange and blue triangles represent mismatches in hDNA; gray octagons correspond to bound MMR proteins; and repaired hDNAs (gene conversion tracts) are highlighted by gray boxes. See text for details.

The model in Figure 7 also incorporates the differential effects of the MMR machinery on CO and NCO events (Welz-Voegele and Jinks-Robertson, 2008). Because the DSBR pathway is associated with the formation of more extensive hDNA at the initial strand-invasion step, the accompanying presence of more mismatches would be more likely trigger MMR-associated antirecombination, reversing the intermediate and thereby precluding CO formation. An intermediate with short hDNA, and relatively few mismatches, would be more likely to evade the MMR machinery, but less likely to mature into an HJ-containing intermediate. The final feature of the model in Figure 7 is that it incorporates the observation that mismatch correction has a strong PCNA requirement, but mitotic antirecombination does not (Stone et al., 2008). We suggest that antirecombination occurs early, before PCNA is loaded to initiate end extension, and involves reversal of intermediate. Mismatch detection that occurs after PCNA engagement could be coupled to DNA synthesis, as it presumably is during DNA replication, or it could occur later during the recombination process, after the interacting duplexes have been resolved. It has been suggested that the interaction of the MMR machinery with PCNA provides strand discrimination information during replication (Umar et al., 1996), and a similar situation might exist during recombination. As shown in Figure 7, if PCNA targets the nascent strand for removal, the genetic result would be gene conversion rather than restoration, as observed here. Not only is the proposed model consistent with observations made using diverse recombination assays in yeast, it makes specific predictions that can be further tested in the gap-repair system.

Conclusions

NCO products of gap repair contain hDNA only in the repaired plasmid and most often on only one side of the gap, consistent with predictions of the SDSA model. In contrast, a bidirectional, trans pattern of hDNA is most often observed in CO products, as predicted by the DSBR model. Data additionally suggest that when an HJ-containing intermediate forms, cleavage of the junctions is highly constrained to generate the CO outcome. Finally, sequence analysis of the gap-repair products demonstrates that CO intermediates contain more extensive hDNA and hence more potential mismatches than do NCO intermediates, providing an explanation for why COs are more efficient targets of MMR-associated antirecombination. Together, these observations suggest a model in which the length of the invading 3′ end determines whether the DSBR or SDSA pathway will be followed, and hence whether a CO or NCO outcome is produced.

EXPERIMENTAL PROCEDURES

Media and growth conditions

Yeast strains were grown nonselectively in YEPD (1% Bacto-yeast extract, 2% Bacto peptone, 2% dextrose) supplemented with 500 μg/ml adenine hemisulfate. Selective growth was on synthetic complete (SC) medium lacking the appropriate nutrient. Ura− segregants were selected on SC plates containing 0.1% 5-flouroorotic acid. All growth was at 30oC.

Gap-repair experiments

Haploid strain SJR1501 and its isogenic mlh1Δ derivative (SJR2157) contain the diverged gap-repair template and have been described previously (Welz-Voegele and Jinks-Robertson, 2008). The fragment for the BglII gap-repair assay was generated by linearizing plasmid pSR840 (Welz-Voegele and Jinks-Robertson, 2008) with BglII, followed by treatment with Mung bean nuclease to remove overhangs. pSR897 was used in BssHII gap-repair experiments and was derived in two steps: the BglII fragment of pSR840 was restored and then a unique BssHII site was introduced by site-directed mutagenesis, replacing 5′-TTTCTGGA with 5′-CGCG (see Figure S1). pSR987 was digested with BssHII prior to transformation of SJR2157.

To distinguish NCO and CO recombinants, His+ colonies were transferred directly to SC-His liquid medium and grown to saturation in 96-well microtiter plates. Cells were diluted 100-fold into nonselective YPD medium, grown again to saturation, and 5 μl of a 1:100 dilution were spotted onto 5-FOA medium. Spots with full growth after two days were scored as NCOs and those with no growth or only a few colonies were scored as COs.

DNA sequence analysis of recombinants

Transformants from the original microtiter plates were diluted into fresh SC-His and grown for 2 days in microtiter plates; all subsequent DNA manipulations were in a 96-well format (http://jinks-robertsonlab.duhs.duke.edu/protocols/yeast_prep.html). DNA was extracted following cell lysis with zymolyase, and appropriate PCR fragments were amplified from total DNA using primers M43, M45, M46 and B32 as appropriate. PCR products were sequenced by the High Throughput Genomic Unit at the University of Washington (Seattle, WA) or by the Duke Comprehensive Cancer Center DNA Analysis Facility using reverse primers M42, M46, R60 and/or R61. Primers sequences are provided in Supplemental Table 1.

Supplementary Material

01

Acknowledgments

We thank lab members and especially Tom Petes for helpful discussions and for comments on the manuscript. This work was supported by a grant from NIH to SJ-R (GM038464).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Allers T, Lichten M. Differential timing and control of noncrossover and crossover recombination during meiosis. Cell. 2001;106:47–57. doi: 10.1016/s0092-8674(01)00416-0. [DOI] [PubMed] [Google Scholar]
  2. Bardwell AJ, Bardwell L, Tomkinson AE, Friedberg EC. Specific cleavage of model recombination and repair intermediates by the yeast Rad1-Rad10 DNA endonuclease. Science. 1994;265:2082–2085. doi: 10.1126/science.8091230. [DOI] [PubMed] [Google Scholar]
  3. Boddy MN, Gaillard PH, McDonald WH, Shanahan P, Yates JR, 3rd, Russell P. Mus81-Eme1 are essential components of a Holliday junction resolvase. Cell. 2001;107:537–548. doi: 10.1016/s0092-8674(01)00536-0. [DOI] [PubMed] [Google Scholar]
  4. Coic E, Gluck L, Fabre F. Evidence for short-patch mismatch repair in Saccharomyces cerevisiae. EMBO J. 2000;19:3408–3417. doi: 10.1093/emboj/19.13.3408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cromie GA, Hyppa RW, Taylor AF, Zakharyevich K, Hunter N, Smith GR. Single Holliday junctions are intermediates of meiotic recombination. Cell. 2006;127:1167–1178. doi: 10.1016/j.cell.2006.09.050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Datta A, Hendrix M, Lipsitch M, Jinks-Robertson S. Dual roles for DNA sequence identity and the mismatch repair system in the regulation of mitotic crossing-over in yeast. Proc Natl Acad Sci USA. 1997;94:9757–9762. doi: 10.1073/pnas.94.18.9757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ferguson DO, Holloman WK. Recombinational repair of gaps in DNA is asymmetric in Ustilago maydis and can be explained by a migrating D-loop model. Proc. Natl. Acad. Sci. USA. 1996;93:5419–5424. doi: 10.1073/pnas.93.11.5419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gilbertson LA, Stahl FW. A test of the double-strand break repair model for meiotic recombination in Saccharomyces cerevisiae. Genetics. 1996;144:27–41. doi: 10.1093/genetics/144.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Harfe BD, Jinks-Robertson S. DNA mismatch repair and genetic instability. Ann Rev Genet. 2000;34:359–399. doi: 10.1146/annurev.genet.34.1.359. [DOI] [PubMed] [Google Scholar]
  10. Ira G, Malkova A, Liberi G, Foiani M, Haber JE. Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell. 2003;115:401–411. doi: 10.1016/s0092-8674(03)00886-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ira G, Satory D, Haber JE. Conservative inheritance of newly synthesized DNA in double-strand break-induced gene conversion. Mol Cell Biol. 2006;26:9424–9429. doi: 10.1128/MCB.01654-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jessop L, Allers T, Lichten M. Infrequent co-conversion of markers flanking a meiotic recombination initiation site in Saccharomyces cerevisiae. Genetics. 2005;169:1353–1367. doi: 10.1534/genetics.104.036509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jinks-Robertson S, Michelitch M, Ramcharan S. Substrate length requirements for efficient mitotic recombination in Saccharomyces cerevisiae. Mol Cell Biol. 1993;13:3937–3950. doi: 10.1128/mcb.13.7.3937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Klein HL, Symington LS. Breaking up just got easier to do. Cell. 2009;138:20–22. doi: 10.1016/j.cell.2009.06.039. [DOI] [PubMed] [Google Scholar]
  15. Kunkel TA, Erie DA. DNA mismatch repair. Annu Rev Biochem. 2005;74:681–710. doi: 10.1146/annurev.biochem.74.082803.133243. [DOI] [PubMed] [Google Scholar]
  16. Kunz C, Fleck O. Role of the DNA repair nucleases Rad13, Rad2 and Uve1 of Schizosaccharomyces pombe in mismatch correction. J Mol Biol. 2001;313:241–253. doi: 10.1006/jmbi.2001.5054. [DOI] [PubMed] [Google Scholar]
  17. Li X, Stith CM, Burgers PM, Heyer WD. PCNA is required for initiation of recombination-associated DNA synthesis by DNA polymerase δ. Mol Cell. 2009;36:704–713. doi: 10.1016/j.molcel.2009.09.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Merker JD, Dominska M, Petes TD. Patterns of heteroduplex formation associated with the initiation of meiotic recombination in the yeast Saccharomyces cerevisiae. Genetics. 2003;165:47–63. doi: 10.1093/genetics/165.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mimitou EP, Symington LS. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature. 2008;455:770–774. doi: 10.1038/nature07312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Myung K, Datta A, Chen C, Kolodner RD. SGS1, the Saccharomyces cerevisiae homologue of BLM and WRN, suppresses genome instability and homeologous recombination. Nature Genet. 2001;27:1–4. doi: 10.1038/83673. [DOI] [PubMed] [Google Scholar]
  21. Nassif N, Penney J, Pal S, Engels WR, Gloor GB. Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair. Mol Cell Biol. 1994;14:1613–1625. doi: 10.1128/mcb.14.3.1613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nicholson A, Hendrix M, Jinks-Robertson S, Crouse GF. Regulation of mitotic homeologous recombination in yeast: functions of mismatch repair and nucleotide excision repair genes. Genetics. 2000;154:133–146. doi: 10.1093/genetics/154.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Osman F, Dixon J, Doe CL, Whitby MC. Generating crossovers by resolution of nicked Holliday junctions: a role for Mus81-Eme1 in meiosis. Mol Cell. 2003;12:761–774. doi: 10.1016/s1097-2765(03)00343-5. [DOI] [PubMed] [Google Scholar]
  24. Palmer S, Schildkraut E, Lazarin R, Nguyen J, Nickoloff JA. Gene conversion tracts in Saccharomyces cerevisiae can be extremely short and highly directional. Nucleic Acids Res. 2003;31:1164–1173. doi: 10.1093/nar/gkg219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Paques F, Haber JE. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 1999;63:349–404. doi: 10.1128/mmbr.63.2.349-404.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Paques F, Leung WY, Haber JE. Expansions and contractions in a tandem repeat induced by double-strand break repair. Mol Cell Biol. 1998;18:2045–2054. doi: 10.1128/mcb.18.4.2045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. San Filippo J, Sung P, Klein H. Mechanism of eukaryotic homologous recombination. Annu Rev Biochem. 2008;77:229–257. doi: 10.1146/annurev.biochem.77.061306.125255. [DOI] [PubMed] [Google Scholar]
  28. Schiestl RH, Prakash S. RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination. Mol Cell Biol. 1988;8:3619–3626. doi: 10.1128/mcb.8.9.3619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sekelsky JJ, McKim KS, Chin GM, Hawley RS. The Drosophila meiotic recombination gene mei-9 encodes a homologue of the yeast excision repair protein Rad1. Genetics. 1995;141:619–627. doi: 10.1093/genetics/141.2.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Spell RM, Jinks-Robertson S. Examination of the roles of the Sgs1 and Srs2 helicases in the enforcement of recombination fidelity in Saccharomyces cerevisiae. Genetics. 2004;168:1855–1865. doi: 10.1534/genetics.104.032771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stone J, Gealy R, Petes T, Jinks-Robertson S. Role of PCNA interactions in the mismatch repair-dependent processing of mitotic and meiotic recombination intermediates in yeast. Genetics. 2008;178:1221–1236. doi: 10.1534/genetics.107.085415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sugawara N, Goldfarb T, Studamire B, Alani E, Haber JE. Heteroduplex rejection during single-strand annealing requires Sgs1 helicase and mismatch repair proteins Msh2 and Msh6 but not Pms1. Proc Natl Acad Sci USA. 2004;101:9315–9320. doi: 10.1073/pnas.0305749101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Surtees JA, Argueso JL, Alani E. Mismatch repair proteins: key regulators of genetic recombination. Cytogenet Genome Res. 2004;107:146–159. doi: 10.1159/000080593. [DOI] [PubMed] [Google Scholar]
  34. Symington LS. Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev. 2002;66:630–670. doi: 10.1128/MMBR.66.4.630-670.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Symington LS, Kang LE, Moreau S. Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae. Nucleic Acids Res. 2000;28:4649–4656. doi: 10.1093/nar/28.23.4649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Szostak JW, Orr-Weaver TL, Rothstein RJ, Stahl FW. The double-strand-break repair model for recombination. Cell. 1983;33:25–35. doi: 10.1016/0092-8674(83)90331-8. [DOI] [PubMed] [Google Scholar]
  37. Tay YD, Sidebotham JM, Wu L. Mph1 requires mismatch repair-independent and -dependent functions of MutSα to regulate crossover formation during homologous recombination repair. Nucleic Acids Res. doi: 10.1093/nar/gkp1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Umar A, Buermeyer AB, Simon JA, Thomas DC, Clark AB, Liskay RM, Kunkel TA. Requirement for PCNA in DNA mismatch repair at a step preceding DNA resynthesis. Cell. 1996;87:65–73. doi: 10.1016/s0092-8674(00)81323-9. [DOI] [PubMed] [Google Scholar]
  39. Welz-Voegele C, Jinks-Robertson S. Sequence divergence impedes crossover more than noncrossover events during mitotic gap repair in yeast. Genetics. 2008;179:1251–1262. doi: 10.1534/genetics.108.090233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhu Z, Chung WH, Shim EY, Lee SE, Ira G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell. 2008;134:981–994. doi: 10.1016/j.cell.2008.08.037. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS190924-supplement-01.doc (92KB, doc)

RESOURCES