Analysis of one-sided marker segregation patterns resulting from mammalian gene targeting

The double-strand break repair (DSBR) model is currently accepted as the paradigm for acts of double-strand break (DSB) repair that lead to crossing over between homologous sequences. The DSBR model predicts that asymmetric heteroduplex DNA (hDNA) will form on both sides of the DSB (two-sided events; 5:3/5:3 segregation). In contrast, in yeast and mammalian cells, a considerable fraction of recombinants are one sided: they display full conversion (6:2 segregation) or half-conversion (5:3 segregation) on one side of the DSB together with normal 4:4 segregation on the other side of the DSB. Two mechanisms have been proposed to account for these observations: (i) hDNA formation is restricted to one side of the DSB or the other, and (ii) recombination is initially two sided, but hDNA repair directed by Holliday junction cuts restores normal 4:4 segregation on that side of the DSB in which the mismatch is closest to the cut junction initiating repair. In this study, we exploited a well-characterized gene-targeting assay to test the predictions that these mechanisms make with respect to the frequency of recombinants displaying 4:4 marker segregation on one side of the DSB. Unexpectedly, the results do not support the predictions of either mechanism. We propose a derivation of mechanism (ii) in which the nicks arising from Holliday junction cleavage are not equivalent with respect to directing repair of adjacent hDNA, possibly as a result of asynchronous cleavage of the DSBR intermediate.     

Evidence for biased holliday junction cleavage and mismatch repair directed by junction cuts during double-strand-break repair in mammalian cells

In mammalian cells, several features of the way homologous recombination occurs between transferred and chromosomal DNA are consistent with the double-strand-break repair (DSBR) model of recombination. In this study, we examined the segregation patterns of small palindrome markers, which frequently escape mismatch repair when encompassed within heteroduplex DNA formed in vivo during mammalian homologous recombination, to test predictions of the DSBR model, in particular as they relate to the mechanism of crossover resolution. According to the canonical DSBR model, crossover between the vector and chromosome results from cleavage of the joint molecule in two alternate sense modes. The two crossover modes lead to different predicted marker configurations in the recombinants, and assuming no bias in the mode of Holliday junction cleavage, the two types of recombinants are expected in equal frequency. However, we propose a revision to the canonical model, as our results suggest that the mode of crossover resolution is biased in favor of cutting the DNA strands upon which DNA synthesis is occurring during formation of the joint molecule. The bias in junction resolution permitted us to examine the potential consequences of mismatch repair acting on the DNA breaks generated by junction cutting. The combination of biased junction resolution with both early and late rounds of mismatch repair can explain the marker patterns in the recombinants.     

The conversion gradient at HIS4 of Saccharomyces cerevisiae. I. Heteroduplex rejection and restoration of Mendelian segregation.

In Saccharomyces cerevisiae, some gene loci manifest gradients in the frequency of aberrant segregation in meiosis, with the high end of each gradient corresponding to a hotspot for DNA double-strand breaks (DSBs). The slope of a gradient is reduced when mismatch repair functions fail to act upon heteroduplex DNA-aberrant segregation frequencies at the low end of the gradient are higher in the absence of mismatch repair. Two models for the role of mismatch repair functions in the generation of meiotic "conversion gradients" have been proposed. The heteroduplex rejection model suggests that recognition of mismatches by mismatch repair enzymes limits hybrid DNA flanking the site of a DSB. The restoration-conversion model proposes that mismatch repair does not affect the length of hybrid DNA, but instead increasingly favors restoration of Mendelian segregation over full conversion with increasing distance from the DSB site. In our experiment designed to distinguish between these two models, data for one subset of well repairable mismatches in the HIS4 gene failed to show restoration-type repair but did indicate reduction in the length of hybrid DNA, supporting the heteroduplex rejection model. However, another subset of data manifested restoration-type repair, indicating a relationship between Holliday junction resolution and mismatch repair. We also present evidence for the infrequent formation of symmetric hybrid DNA during meiotic DSB repair.     

Testing predictions of the double-strand break repair model relating to crossing over in Mammalian cells

In yeast, four-stranded, biparental "joint molecules" containing a pair of Holliday junctions are demonstrated intermediates in the repair of meiotic double-strand breaks (DSBs). Genetic and physical evidence suggests that when joint molecules are resolved by the cutting of each of the two Holliday junctions, crossover products result at least most of the time. The double-strand break repair (DSBR) model is currently accepted as a paradigm for acts of DSB repair that lead to crossing over. In this study, a well-defined mammalian gene-targeting assay was used to test predictions that the DSBR model makes about the frequency and position of hDNA in recombinants generated by crossing over. The DSBR model predicts that hDNA will frequently form on opposite sides of the DSB in the two homologous sequences undergoing recombination [half conversion (HC); 5:3, 5:3 segregation]. By examining the segregation patterns of poorly repairable small palindrome genetic markers, we show that this configuration of hDNA is rare. Instead, in a large number of recombinants, full conversion (FC) events in the direction of the unbroken chromosomal sequence (6:2 segregation) were observed on one side of the DSB. A conspicuous fraction of the unidirectional FC events was associated with normal 4:4 marker segregation on the other side of the DSB. In addition, a large number of recombinants displayed evidence of hDNA formation. In several, hDNA was symmetrical on one side of the DSB, suggesting that the two homologous regions undergoing recombination swapped single strands of the same polarity. These data are considered within the context of modified versions of the DSBR model.     

A Test of the Double-Strand Break Repair Model for Meiotic Recombination in Saccharomyces Cerevisiae

We tested predictions of the double-strand break repair (DSBR) model for meiotic recombination by examining the segregation patterns of small palindromic insertions, which frequently escape mismatch repair when in heteroduplex DNA. The palindromes flanked a well characterized DSB site at the ARG4 locus. The ``canonical'''' DSBR model, in which only 5'' ends are degraded and resolution of the four-stranded intermediate is by Holliday junction resolvase, predicts that hDNA will frequently occur on both participating chromatids in a single event. Tetrads reflecting this configuration of hDNA were rare. In addition, a class of tetrads not predicted by the canonical DSBR model was identified. This class represented events that produced hDNA in a ``trans'''' configuration, on opposite strands of the same duplex on the two sides of the DSB site. Whereas most classes of convertant tetrads had typical frequencies of associated crossovers, tetrads with trans hDNA were parental for flanking markers. Modified versions of the DSBR model, including one that uses a topoisomerase to resolve the canonical DSBR intermediate, are supported by these data.     

Systematic mutagenesis of the Saccharomyces cerevisiae MLH1 gene reveals distinct roles for Mlh1p in meiotic crossing over and in vegetative and meiotic mismatch repair

In eukaryotic cells, DNA mismatch repair is initiated by a conserved family of MutS (Msh) and MutL (Mlh) homolog proteins. Mlh1 is unique among Mlh proteins because it is required in mismatch repair and for wild-type levels of crossing over during meiosis. In this study, 60 new alleles of MLH1 were examined for defects in vegetative and meiotic mismatch repair as well as in meiotic crossing over. Four alleles predicted to disrupt the Mlh1p ATPase activity conferred defects in all functions assayed. Three mutations, mlh1-2, -29, and -31, caused defects in mismatch repair during vegetative growth but allowed nearly wild-type levels of meiotic crossing over and spore viability. Surprisingly, these mutants did not accumulate high levels of postmeiotic segregation at the ARG4 recombination hotspot. In biochemical assays, Pms1p failed to copurify with mlh1-2, and two-hybrid studies indicated that this allele did not interact with Pms1p and Mlh3p but maintained wild-type interactions with Exo1p and Sgs1p. mlh1-29 and mlh1-31 did not alter the ability of Mlh1p-Pms1p to form a ternary complex with a mismatch substrate and Msh2p-Msh6p, suggesting that the region mutated in these alleles could be responsible for signaling events that take place after ternary complex formation. These results indicate that mismatches formed during genetic recombination are processed differently than during replication and that, compared to mismatch repair functions, the meiotic crossing-over role of MLH1 appears to be more resistant to mutagenesis, perhaps indicating a structural role for Mlh1p during crossing over.     

Meiotic versus mitotic recombination: two different routes for double-strand break repair: the different functions of meiotic versus mitotic DSB repair are reflected in different pathway usage and different outcomes

Studies in the yeast Saccharomyces cerevisiae have validated the major features of the double-strand break repair (DSBR) model as an accurate representation of the pathway through which meiotic crossovers (COs) are produced. This success has led to this model being invoked to explain double-strand break (DSB) repair in other contexts. However, most non-crossover (NCO) recombinants generated during S. cerevisiae meiosis do not arise via a DSBR pathway. Furthermore, it is becoming increasingly clear that DSBR is a minor pathway for recombinational repair of DSBs that occur in mitotically-proliferating cells and that the synthesis-dependent strand annealing (SDSA) model appears to describe mitotic DSB repair more accurately. Fundamental dissimilarities between meiotic and mitotic recombination are not unexpected, since meiotic recombination serves a very different purpose (accurate chromosome segregation, which requires COs) than mitotic recombination (repair of DNA damage, which typically generates NCOs).     

Repair of specific base pair mismatches formed during meiotic recombination in the yeast Saccharomyces cerevisiae.

Heteroduplexes formed between DNA strands derived from different homologous chromosomes are an intermediate in meiotic crossing over in the yeast Saccharomyces cerevisiae and other eucaryotes. A heteroduplex formed between wild-type and mutant genes will contain a base pair mismatch; failure to repair this mismatch will lead to postmeiotic segregation (PMS). By analyzing the frequency of PMS for various mutant alleles in the yeast HIS4 gene, we showed that C/C mismatches were inefficiently repaired relative to all other point mismatches. These other mismatches (G/G, G/A, T/T, A/A, T/C, C/A, A/A, and T/G) were repaired with approximately the same efficiency. We found that in spores with unrepaired mismatches in heteroduplexes, the nontranscribed strand of the HIS4 gene was more frequently donated than the transcribed strand. In addition, the direction of repair for certain mismatches was nonrandom.     

The Saccharomyces cerevisiae Msh2 mismatch repair protein localizes to recombination intermediates in vivo

Mismatch repair proteins act during double-strand break repair (DSBR) to correct mismatches in heteroduplex DNA, to suppress recombination between divergent sequences, and to promote removal of nonhomologous DNA at DSB ends. We investigated yeast Msh2p association with recombination intermediates in vivo using chromatin immunoprecipitation. During DSBR involving nonhomologous ends, Msh2p localized strongly to recipient and donor sequences. Localization required Msh3p and was greatly reduced in rad50delta strains. Minimal localization of Msh2p was observed during fully homologous repair, but this was increased in rad52delta strains. These findings argue that Msh2p-Msh3p associates with intermediates early in DSBR to participate in the rejection of homeologous pairing and to stabilize nonhomologous tails for cleavage by Rad1p-Rad10p endonuclease.     

Mechanisms of double-strand-break repair during gene targeting in mammalian cells

In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.     

Biased short tract repair of palindromic loop mismatches in mammalian cells.

Mismatch repair of palindromic loops in the presence or absence of single-base mismatches was investigated in wild-type and mismatch-binding defective mutant Chinese hamster ovary cells. Recombination intermediates with a maximum heteroduplex DNA (hDNA) region of 697 bp contained a centrally located, phenotypically silent 12-base palindromic loop mismatch, and/or five single-base mismatches. In wild-type cells, both loops and single-base mismatches were efficiently repaired (80-100%). When no other mismatches were present in hDNA, loops were retained with a 1.6-1.9:1 bias. However, this bias was eliminated when single-base mismatches were present, perhaps because single-base mismatches signal nick-directed repair. In the multiple marker crosses, most repair tracts were long and continuous, with preferential loss of markers in cis to proximal nicks, consistent with nicks directing most repair in this situation. However, approximately 25% of repair tracts were discontinuous as a result of loop-specific repair, or from segregation or short tract repair of single-base mismatches. In mutant cells, single-base mismatches were repaired less frequently, but the loop was still repaired efficiently and with bias toward loop retention, indicating that the defect in these cells does not affect loop-specific repair. Repair tracts in products from mutant cells showed a wide variety of mosaic patterns reflecting short regions of repair and segregation consistent with reduced nick-directed repair. In mutant cells, single-base mismatches were repaired more efficiently in the presence of the loop than in its absence, a likely consequence of corepair initiated at the loop.     

Tests of the Double-Strand-Break Repair Model for Red-Mediated Recombination of Phage λ and Plasmid λdv

The double-strand-break repair (DSBR) model was formulated to account for various aspects of yeast mitotic and meiotic recombination. In this study three features of the DSBR model are tested for Red-mediated recombination between phage lambda and lambda dv, a plasmid that is perfectly homologous to about 10% of lambda. The results support the applicability of the DSBR model to lambda's Red system: (1) Creating a double-strand-break (DSB) within the region of homology shared by phage and plasmid increases their genetic interaction by about 20-fold. A DSB outside the region of shared homology has no such effect. (2) Both patches, i.e., simple marker rescue, and splices, i.e., co-integration of the phage and plasmid, are stimulated by a DSB in the region of shared homology. (3) Co-integrants harbor a duplication of the region of shared homology. Among co-integrants that were formed by the creation of a DSB, there is a preferential loss of whichever allele was in cis to a utilized cut site. The DSBR model as originally formulated involves the isomerization and cleavage of Holliday junctions to resolve the canonical intermediate. We propose as an alternative mechanism that a topoisomerase can resolve the canonical DSBR intermediate.     

Control of crossing over

The Holliday junction is a central intermediate in homologous recombination. It consists of a four-way structure that can be resolved by cleavage to give either the crossover or noncrossover products observed. We show here that the formation of these products is controlled by the E. coli resolvasome (RuvABC) in such way that double-strand break repair (DSBR) leads to crossing over and single-strand gap repair (SSGR) does not lead to crossing over. We argue that the positioning of the RuvABC complex and its consequent direction of junction-cleavage is not random. In fact, the action of the RuvABC complex avoids crossing over in the most commonly predicted situations where Holliday junctions are encountered in DNA replication and repair. Our observations suggest that the positioning of the resolvasome may provide a general biochemical mechanism by which cells can control crossing over in recombination.     

Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events.

In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells.     

Efficient incorporation of large (>2 kb) heterologies into heteroduplex DNA: Pms1/Msh2-dependent and -independent large loop mismatch repair in Saccharomyces cerevisiae

DNA double-strand break (DSB) repair in yeast is effected primarily by gene conversion. Conversion can conceivably result from gap repair or from mismatch repair of heteroduplex DNA (hDNA) in recombination intermediates. Mismatch repair is normally very efficient, but unrepaired mismatches segregate in the next cell division, producing sectored colonies. Conversion of small heterologies (single-base differences or insertions <15 bp) in meiosis and mitosis involves mismatch repair of hDNA. The repair of larger loop mismatches in plasmid substrates or arising by replication slippage is inefficient and/or independent of Pms1p/Msh2p-dependent mismatch repair. However, large insertions convert readily (without sectoring) during meiotic recombination, raising the question of whether large insertions convert by repair of large loop mismatches or by gap repair. We show that insertions of 2.2 and 2.6 kbp convert efficiently during DSB-induced mitotic recombination, primarily by Msh2p- and Pms1p-dependent repair of large loop mismatches. These results support models in which Rad51p readily incorporates large heterologies into hDNA. We also show that large heterologies convert more frequently than small heterologies located the same distance from an initiating DSB and propose that this reflects Msh2-independent large loop-specific mismatch repair biased toward loop loss.     

Coordination of DNA ends during double-strand-break repair in bacteriophage T4

The extensive chromosome replication (ECR) model of double-strand-break repair (DSBR) proposes that each end of a double-strand break (DSB) is repaired independently by initiating extensive semiconservative DNA replication after strand invasion into homologous template DNA. In contrast, several other DSBR models propose that the two ends of a break are repaired in a coordinated manner using a single repair template with only limited DNA synthesis. We have developed plasmid and chromosomal recombinational repair assays to assess coordination of the broken ends during DSBR in bacteriophage T4. Results from the plasmid assay demonstrate that the two ends of a DSB can be repaired independently using homologous regions on two different plasmids and that extensive replication is triggered in the process. These findings are consistent with the ECR model of DSBR. However, results from the chromosomal assay imply that the two ends of a DSB utilize the same homologous repair template even when many potential templates are present, suggesting coordination of the broken ends during chromosomal repair. This result is consistent with several coordinated models of DSBR, including a modified version of the ECR model.     

Double-strand break repair plays a role in repeat instability in a fragile X mouse model

Expansion of a CGG-repeat tract in the 5′ UTR of FMR1 is responsible for the Fragile X-related disorders (FXDs), FXTAS, FXPOI and FXS. Previous work in a mouse model of these disorders has implicated proteins in the base excision and the mismatch repair (MMR) pathways in the expansion mechanism. However, the precise role of these factors in this process is not well understood. The essential role of MutLγ, a complex that plays a minor role in MMR but that is essential for resolving Holliday junctions during meiosis, raises the possibility that expansions proceed via a Holliday junction-like intermediate that is processed to generate a double-strand break (DSB). We show here in an FXD mouse model that LIG4, a ligase essential for non-homologous end-joining (NHEJ), a form of DSB repair (DSBR), protects against expansions. However, a mutation in MRE11, a nuclease that is important for several other DSBR pathways including homologous recombination (HR), has no effect on the extent of expansion. Our results suggest that the expansion pathway competes with NHEJ for the processing of a DSB intermediate. Thus, expansion likely proceeds via an NHEJ-independent DSBR pathway that may also be HR-independent.     

Regulation of Msh4-Msh5 association with meiotic chromosomes in budding yeast

In the baker’s yeast Saccharomyces cerevisiae, most of the meiotic crossovers are generated through a pathway involving the highly conserved mismatch repair related Msh4-Msh5 complex. To understand the role of Msh4-Msh5 in meiotic crossing over, we determined its genome wide in vivo binding sites in meiotic cells. We show that Msh5 specifically associates with DSB hotspots, chromosome axes, and centromeres on chromosomes. A basal level of Msh5 association with these chromosomal features is observed even in the absence of DSB formation (spo11Δ mutant) at the early stages of meiosis. But efficient binding to DSB hotspots and chromosome axes requires DSB formation and resection and is enhanced by double Holliday junction structures. Msh5 binding is also correlated to DSB frequency and enhanced on small chromosomes with higher DSB and crossover density. The axis protein Red1 is required for Msh5 association with the chromosome axes and DSB hotspots but not centromeres. Although binding sites of Msh5 and other pro-crossover factors like Zip3 show extensive overlap, Msh5 associates with centromeres independent of Zip3. These results on Msh5 localization in wild type and meiotic mutants have implications for how Msh4-Msh5 works with other pro-crossover factors to ensure crossover formation.     

Formation and repair of heteroduplex DNA on both sides of the double-strand break during mammalian gene targeting

In this study, we examined homologous recombination in mammalian cells using a gene targeting assay in which the introduction of a double-strand-break (DSB) in the vector-borne region of homology to the chromosome resulted in targeted vector integration. The vector-borne DSB was flanked with small palindromic insertions that, when encompassed within heteroduplex DNA (hDNA) formed during targeted vector integration, were capable of avoiding the activity of the mismatch repair (MMR) system. When used in conjunction with an isolation procedure in which the product(s) of each targeted vector integration event were retained for molecular analysis, information about recombination mechanisms was obtained. The examination of marker segregation patterns in independent recombinants revealed the following, (i) hDNA tracts could form simultaneously on each side of the DSB and in both participating homologous regions. Clonal analysis of sectored recombinants revealed that, in the homologous repeats generated by the recombination event, vector-borne palindrome and chromosomal markers were linked in the expected way in each strand of the hDNA intermediate, (ii) hDNA tracts were subject to MMR processing that occurred on opposite sides of the DSB, and (iii) in the majority of recombinants, the vector-borne marker was replaced with the corresponding marker from the chromosome. Bidirectional hDNA formation and MMR processing of both sides of the DSB are consistent with the double-strand-break repair (DSBR) model of recombination.     

Patterns of heteroduplex formation associated with the initiation of meiotic recombination in the yeast Saccharomyces cerevisiae

The double-strand break repair (DSBR) model of recombination predicts that heteroduplexes will be formed in regions that flank the double-strand break (DSB) site and that the resulting intermediate is resolved to generate either crossovers or noncrossovers for flanking markers. Previous studies in Saccharomyces cerevisiae, however, failed to detect heteroduplexes on both sides of the DSB site. Recent physical studies suggest that some recombination events involve heterodupex formation by a mechanism, synthesis-dependent strand annealing (SDSA), that is inherently asymmetric with respect to the DSB site and that leads exclusively to noncrossovers of flanking markers. Below, we demonstrate that many of the recombination events initiated at the HIS4 recombination hotspot are consistent with a variant of the DSBR model in which the extent of heteroduplex on one side of the DSB site is much greater than that on the other. Events that include only one flanking marker in the heteroduplex (unidirectional events) are usually resolved as noncrossovers, whereas events that include both flanking markers (bidirectional events) are usually resolved as crossovers. The unidirectional events may represent SDSA, consistent with the conclusions of others, although other possibilities are not excluded. We also show that the level of recombination reflects the integration of events initiated at several different DSB sites, and we identify a subset of gene conversion events that may involve break-induced replication (BIR) or repair of a double-stranded DNA gap.