Differential timing and control of noncrossover and crossover recombination during meiosis.

Unitary models of meiotic recombination postulate that a central intermediate containing Holliday junctions is resolved to generate either noncrossover or crossover recombinants, both of which contain heteroduplex DNA. Contrary to this expectation, we find that during meiosis in Saccharomyces cerevisiae, noncrossover heteroduplex products are formed at the same time as Holliday junction intermediates. Crossovers appear later, when these intermediates are resolved. Furthermore, noncrossover and crossover recombination are regulated differently. ndt80 mutants arrest in meiosis with unresolved Holliday junction intermediates and very few crossovers, while noncrossover heteroduplex products are formed at normal levels and with normal timing. These results suggest that crossovers are formed by resolution of Holliday junction intermediates, while most noncrossover recombinants arise by a different, earlier pathway.     

Saccharomyces cerevisiae Mer3 is a DNA helicase involved in meiotic crossing over

Crossing over is regulated to occur at least once per each pair of homologous chromosomes during meiotic prophase to ensure proper segregation of chromosomes at the first meiotic division. In a mer3 deletion mutant of Saccharomyces cerevisiae, crossing over is decreased, and the distribution of the crossovers that occur is random. The predicted Mer3 protein contains seven motifs characteristic of the DExH box type of DNA/RNA helicases. The mer3G166D and the mer3K167A mutation, amino acid substitutions of conserved residues in a putative nucleotide-binding domain of the helicase motifs caused a defect in the transition of meiosis-specific double-strand breaks to later intermediates, decreased crossing over, and reduced crossover interference. The purified Mer3 protein was found to have DNA helicase activity. This helicase activity was reduced by the mer3GD mutation to <1% of the wild-type activity, even though binding of the mutant protein to single- and double-strand DNA was unaffected. The mer3KA mutation eliminated the ATPase activity of the wild-type protein. These results demonstrate that Mer3 is a DNA helicase that functions in meiotic crossing over.     

Mnd1/Hop2 facilitates Dmc1-dependent interhomolog crossover formation in meiosis of budding yeast

During meiosis, each chromosome must pair with its homolog and undergo meiotic crossover recombination in order to segregate properly at the first meiotic division. Recombination in meiosis in Saccharomyces cerevisiae relies on two Escherichia coli recA homologs, Rad51 and Dmc1, as well as the more recently discovered heterodimer Mnd1/Hop2. Meiotic recombination in S. cerevisiae mnd1 and hop2 single mutants is initiated via double-strand breaks (DSBs) but does not progress beyond this stage; heteroduplex DNA, joint molecules, and crossovers are not detected. Whereas hop2 and mnd1 single mutants are profoundly recombination defective, we show that mnd1 rad51, hop2 rad51, and mnd1 rad17 double mutants are able to carry out crossover recombination. Interestingly, noncrossover recombination is absent, indicating a role for Mnd1/Hop2 in the designation of DSBs for noncrossover recombination. We demonstrate that in the rad51 mnd1 double mutant, recombination is more likely to occur between repetitive sequences on nonhomologous chromosomes. Our results support a model in which Mnd1/Hop2 is required for DNA-DNA interactions that help ensure Dmc1-mediated stable strand invasion between homologous chromosomes, thereby preserving genomic integrity.     

Heteroduplex DNA in meiotic recombination in Drosophila mei-9 mutants

Meiotic recombination gives rise to crossovers, which are required in most organisms for the faithful segregation of homologous chromosomes during meiotic cell division. Characterization of crossover-defective mutants has contributed much to our understanding of the molecular mechanism of crossover formation. We report here a molecular analysis of recombination in a Drosophila melanogaster crossover-defective mutant, mei-9. In the absence of mei-9 activity, postmeiotic segregation associated with noncrossovers occurs at the expense of crossover products, suggesting that the underlying meiotic function for MEI-9 is in crossover formation rather than mismatch repair. In support of this, analysis of the arrangement of heteroduplex DNA in the postmeiotic segregation products reveals different patterns from those observed in Drosophila Msh6 mutants, which are mismatch-repair defective. This analysis also provides evidence that the double-strand break repair model applies to meiotic recombination in Drosophila. Our results support a model in which MEI-9 nicks Holliday junctions to generate crossovers during meiotic recombination, and, in the absence of MEI-9 activity, the double Holliday junction intermediate instead undergoes dissolution to generate noncrossover products in which heteroduplex is unrepaired.     

The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length

Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLβ heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLβ recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLβ ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLβ may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1’s activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLβ ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.     

Eliminating Both Canonical and Short-Patch Mismatch Repair in Drosophila melanogaster Suggests a New Meiotic Recombination Model

In most meiotic systems, recombination is essential to form connections between homologs that ensure their accurate segregation from one another. Meiotic recombination is initiated by DNA double-strand breaks that are repaired using the homologous chromosome as a template. Studies of recombination in budding yeast have led to a model in which most early repair intermediates are disassembled to produce noncrossovers. Selected repair events are stabilized so they can proceed to form double-Holliday junction (dHJ) intermediates, which are subsequently resolved into crossovers. This model is supported in yeast by physical isolation of recombination intermediates, but the extent to which it pertains to animals is unknown. We sought to test this model in Drosophila melanogaster by analyzing patterns of heteroduplex DNA (hDNA) in recombination products. Previous attempts to do this have relied on knocking out the canonical mismatch repair (MMR) pathway, but in both yeast and Drosophila the resulting recombination products are complex and difficult to interpret. We show that, in Drosophila, this complexity results from a secondary, short-patch MMR pathway that requires nucleotide excision repair. Knocking out both canonical and short-patch MMR reveals hDNA patterns that reveal that many noncrossovers arise after both ends of the break have engaged with the homolog. Patterns of hDNA in crossovers could be explained by biased resolution of a dHJ; however, considering the noncrossover and crossover results together suggests a model in which a two-end engagement intermediate with unligated HJs can be disassembled by a helicase to a produce noncrossover or nicked by a nuclease to produce a crossover. While some aspects of this model are similar to the model from budding yeast, production of both noncrossovers and crossovers from a single, late intermediate is a fundamental difference that has important implications for crossover control.     

Primary products of break-induced recombination by Escherichia coli RecE pathway.

Alternative models for break-induced recombination predict different distributions of primary products. The double-stranded break-repair model predicts a noncrossover product and equimolar amounts of two crossover products. The one-end pairing model predicts two crossover products, but not necessarily in equimolar amounts, and the single-stranded annealing model predicts deletion of the fragment between the pairing sequences. Depending on the structure of the recombining substrate(s) and the nature of the resectioning step that precedes strand annealing, the single-stranded annealing mechanism would yield only one or both crossover products. We tested these predictions for the RecE recombination pathway of Escherichia coli. Nonreplicating intramolecular recombination substrates with a double-stranded break (DSB) within one copy of a direct repeat were released from chimera lambda phage by in vivo restriction, and the distribution of primary circular recombination products was determined. Noncrossover products were barely detectable, and the molar ratio of the two crossover products was proportional to the length ratio of the homologous ends flanking the DSB. These results suggest an independent pairing of each end with the intact homolog and argue against the double-stranded break-repair model. However, the results do not distinguish alternative pairing mechanisms (strand invasion and strand annealing). The kinetics of heteroduplex formation and heteroduplex strand polarity were investigated. Immediately following the DSB induction, heteroduplex formation was done by pairing the strands ending 3' at the break. A slow accumulation of the complementary heteroduplex made by the pairing of the strands ending 5' at the break (5' heteroduplexes) was observed at a larger stage. The observed bias in heteroduplex strand polarity depended on DSB induction at a specific site. The 5' heteroduplexes may have been generated by reciprocal strand exchange, pairing that is not strand specific, or strand-specific pairing induced at random breaks.     

Trans events associated with crossovers are revealed in the absence of mismatch repair genes in Saccharomyces cerevisiae

Genetic analysis of recombination in Saccharomyces cerevisiae has revealed products with structures not predicted by the double-strand break repair model of meiotic recombination. A particular type of recombinant containing trans heteroduplex DNA has been observed at two loci. Trans events were originally identified only in tetrads in which the non-Mendelian segregations were not associated with a crossover. Because of this, these events were proposed to have arisen from the unwinding of double Holliday junctions. Previous studies used palindromes, refractory to mismatch repair, as genetic markers whereas we have used a complementary approach of deleting mismatch repair proteins to identify heteroduplex DNA. We found that the markers occurred in trans and were associated with crossovers. In both mlh1Delta and msh2Delta strains, the frequency of trans events associated with a crossover exceeded that predicted from the random association of crossovers with noncrossover trans events. We propose two different models to account for trans events associated with crossovers and discuss the relevance to wild-type DSB repair.     

Meiotic chromosome synapsis-promoting proteins antagonize the anti-crossover activity of sgs1

Sgs1, the budding yeast homolog of the mammalian BLM helicase, has been implicated in preventing excess recombination during both vegetative growth and meiosis. Most meiotic crossover (CO) recombination requires full function of a set of yeast proteins (Zip1, Zip2, Zip3, Zip4/Spo22, Mer3, Msh4, and Msh5, termed the SIC or ZMM proteins) that are also required for homologous chromosome synapsis. We report here genetic and molecular assays showing that sgs1 single mutants display relatively modest increases in CO recombination (less than 1.6-fold relative to wild-type). In contrast, a much greater CO increase is seen when an sgs1 mutation is introduced into the CO- and synapsis-deficient zip1, zip2, zip3, mer3, or msh4 mutants (2- to 8-fold increase). Furthermore, close juxtaposition of the axes of homologous chromosomes is restored. CO restoration in the mutants is not accompanied by significant changes in noncrossover (NCO) recombinant frequencies. These findings show that Sgs1 has potent meiotic anti-CO activity, which is normally antagonized by SIC/ZMM proteins. Our data reinforce previous proposals for an early separation of meiotic processes that form CO and NCO recombinants.     

The MER3 helicase involved in meiotic crossing over is stimulated by single-stranded DNA-binding proteins and unwinds DNA in the 3' to 5' direction

The meiosis-specific MER3 protein of Saccharomyces cerevisiae is required for crossing over, which ensures faithful segregation of homologous chromosomes at the first meiotic division. The predicted sequence of the MER3 protein contains the seven motifs characteristic of the DExH-box type of DNA/RNA helicases. The purified MER3 protein is a DNA helicase, which can displace a 50-nucleotide fragment annealed to a single-stranded circular DNA. MER3 was found to have ATPase activity, which was stimulated either by single- or double-stranded DNA. The turnover rate, k(cat), of ATP hydrolysis was approximately 500/min in the presence of either DNA. MER3 was able to efficiently displace relatively long 631-nucleotide fragments from single-stranded circular DNA only in the presence of the S. cerevisiae single-stranded DNA-binding protein, RPA (replication protein A). It appears that RPA inhibits re-annealing of the single-stranded products of the MER3 helicase. The MER3 helicase was found to unwind DNA in the 3' to 5' direction relative to single-stranded regions in the DNA substrates. Possible roles for the MER3 helicase in meiotic crossing over are discussed.     

The MER3 DNA helicase catalyzes the unwinding of holliday junctions

The MER3 protein of Saccharomyces cerevisiae is required for crossover in meiosis and has been suggested to act at the initiation of homologous pairing and the resolution of Holliday junctions. The purified MER3 protein is a DNA helicase that translocates along single-stranded DNA in the 3' to 5' direction displacing annealed DNA fragments. Here, MER3 was found to be able to unwind various double-stranded DNA (dsDNA) substrates, including a 30-bp dsDNA with a 20-nucleotide 3'-overhang, a 30-bp dsDNA with a 20-nucleotide 5'-overhang, a 50-bp dsDNA with blunt ends, and a Holliday junction with 25-bp arms, each of which had a blunt end. Efficient unwinding of the 3'-overhang substrate appeared to initiate by the binding of MER3 to the 3' single-stranded tail in a reaction that required six or more unpaired bases. Unwinding of the blunt end and 5'-overhang substrates appeared to initiate at the blunt ends of these substrates. Unwinding of the Holliday junction was more efficient than the unwinding of the blunt and 5'-overhang substrates and was influenced by Mg(2+) concentrations that cause changes in the structure of the junction. Possible roles for Holliday junction unwinding in meiotic crossover are discussed.     

Human DNA Helicase B Functions in Cellular Homologous Recombination and Stimulates Rad51-Mediated 5′-3′ Heteroduplex Extension In Vitro

Homologous recombination is involved in the repair of DNA damage and collapsed replication fork, and is critical for the maintenance of genomic stability. Its process involves a network of proteins with different enzymatic activities. Human DNA helicase B (HDHB) is a robust 5′-3′ DNA helicase which accumulates on chromatin in cells exposed to DNA damage. HDHB facilitates cellular recovery from replication stress, but its role in DNA damage response remains unclear. Here we report that HDHB silencing results in reduced sister chromatid exchange, impaired homologous recombination repair, and delayed RPA late-stage foci formation induced by ionizing radiation. Ectopically expressed HDHB colocalizes with Rad51, Rad52, RPA, and ssDNA. In vitro, HDHB stimulates Rad51-mediated heteroduplex extension in 5′-3′ direction. A helicase-defective mutant HDHB failed to promote this reaction. Our studies implicate HDHB promotes homologous recombination in vivo and stimulates 5′-3′ heteroduplex extension during Rad51-mediated strand exchange in vitro.     

Crossover/noncrossover differentiation, synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene transition of meiosis

Yeast mutants lacking meiotic proteins Zip1, Zip2, Zip3, Mer3, and/or Msh5 (ZMMs) were analyzed for recombination, synaptonemal complex (SC), and meiotic progression. At 33 degrees C, recombination-initiating double-strand breaks (DSBs) and noncrossover products (NCRs) form normally while formation of single-end invasion strand exchange intermediates (SEIs), double Holliday junctions, crossover products (CRs), and SC are coordinately defective. Thus, during wild-type meiosis, recombinational interactions are differentiated into CR and NCR types very early, prior to onset of stable strand exchange and independent of SC. By implication, crossover interference does not require SC formation. We suggest that SC formation may require interference. Subsequently, CR-designated DSBs undergo a tightly coupled, ZMM-promoted transition that yields SEI-containing recombination complexes embedded in patches of SC. zmm mutant phenotypes differ strikingly at 33 degrees C and 23 degrees C, implicating higher temperature as a positive effector of recombination and identifying a checkpoint that monitors local CR-specific events, not SC formation, at late leptotene.     

Analysis of a Gene Conversion Gradient at the His4 Locus in Saccharomyces Cerevisiae

Heteroduplexes formed between genes on homologous chromosomes are intermediates in meiotic recombination. In the HIS4 gene of Saccharomyces cerevisiae, most mutant alleles at the 5' end of the gene have a higher rate of meiotic recombination (gene conversion) than mutant alleles at the 3' end of the gene. Such gradients are usually interpreted as indicating a higher frequency of heteroduplex formation at the high conversion end of the gene. We present evidence indicating that the gradient of conversion at HIS4 primarily reflects the direction of mismatch repair rather than the frequency of heteroduplex formation. We also identify a site located between the 5' end of HIS4 and the 3' end of BIK1 that stimulates heteroduplex formation at HIS4 and BIK1.     

Decreased meiotic intergenic recombination and increased meiosis I nondisjunction in exo1 mutants of Saccharomyces cerevisiae

Exonuclease I was originally identified as a 5' --> 3' deoxyribonuclease present in fractionated extracts of Schizosaccharomyces pombe and Saccharomyces cerevisiae. Genetic analysis of exo1 mutants of both yeasts revealed no major defect in meiosis, suggesting that exonuclease I is unlikely to be the primary activity that processes meiosis-specific double-strand breaks (DSBs). We report here that exo1 mutants of S. cerevisiae exhibit subtle but complex defects in meiosis. Diploids containing a homozygous deletion of EXO1 show decreased spore viability associated with an increase in meiosis I nondisjunction, while intergenic recombination is reduced about twofold. Exo1p functions in the same pathway as Msh5p for intergenic recombination. The length of heteroduplex tracts within the HIS4 gene is unaffected by the exo1 mutation. These results suggest that Exo1p is unlikely to play a major role in processing DSBs to form single-stranded tails at HIS4, but instead appears to promote crossing over to ensure disjunction of homologous chromosomes. In addition, our data indicate that exonuclease I may have a minor role in the correction of large DNA mismatches that occur in heteroduplex DNA during meiotic recombination at the HIS4 locus.     

Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast

Very few gene conversions in mitotic cells are associated with crossovers, suggesting that these events are regulated. This may be important for the maintenance of genetic stability. We have analyzed the relationship between homologous recombination and crossing-over in haploid budding yeast and identified factors involved in the regulation of crossover outcomes. Gene conversions unaccompanied by a crossover appear 30 min before conversions accompanied by exchange, indicating that there are two different repair mechanisms in mitotic cells. Crossovers are rare (5%), but deleting the BLM/WRN homolog, SGS1, or the SRS2 helicase increases crossovers 2- to 3-fold. Overexpressing SRS2 nearly eliminates crossovers, whereas overexpression of RAD51 in srs2Delta cells almost completely eliminates the noncrossover recombination pathway. We suggest Sgs1 and its associated topoisomerase Top3 remove double Holliday junction intermediates from a crossover-producing repair pathway, thereby reducing crossovers. Srs2 promotes the noncrossover synthesis-dependent strand-annealing (SDSA) pathway, apparently by regulating Rad51 binding during strand exchange.     

Roles of exonucleases and translesion synthesis DNA polymerases during mitotic gap repair in yeast

Transformation-based gap-repair assays have long been used to model the repair of mitotic double-strand breaks (DSBs) by homologous recombination in yeast. In the current study, we examine genetic requirements of two key processes involved in DSB repair: (1) the processive 5′-end resection that is required to efficiently engage a repair template and (2) the filling of resected ends by DNA polymerases. The specific gap-repair assay used allows repair events resolved as crossover versus noncrossover products to be distinguished, as well as the extent of heteroduplex DNA formed during recombination to be measured. To examine end resection, the efficiency and outcome of gap repair were monitored in the absence of the Exo1 exonuclease and the Sgs1 helicase. We found that either Exo1 or Sgs1 presence is sufficient to inhibit gap-repair efficiency over 10-fold, consistent with resection-mediated destruction of the introduced plasmid. In terms of DNA polymerase requirements for gap repair, we focused specifically on potential roles of the Pol ζ and Pol η translesion synthesis DNA polymerases. We found that both Pol ζ and Pol η are necessary for efficient gap repair and that each functions independently of the other. These polymerases may be involved either in the initiation of DNA synthesis from the an invading end, or in a gap-filling process that is required to complete recombination.     

Mechanistic analysis of a DNA end processing pathway mediated by the Xenopus Werner syndrome protein

The first step of homology-dependent repair of DNA double-strand breaks is the strand-specific processing of DNA ends to generate 3' single-strand tails. Despite its importance, the molecular mechanism underlying end processing is poorly understood in eukaryotic cells. We have taken a biochemical approach to investigate DNA end processing in nucleoplasmic extracts derived from the unfertilized eggs of Xenopus laevis. We found that double-strand DNA ends are specifically degraded in the 5' --> 3' direction in this system. The reaction consists of two steps: an ATP-dependent unwinding of double-strand ends and an ATP-independent 5' --> 3' degradation of single-strand tails. We also found that the Xenopus Werner syndrome protein, a member of the RecQ helicase family, plays an important role in DNA end processing. Mechanistically, Xenopus Werner syndrome protein (xWRN) is required for the unwinding of DNA ends but not for the degradation of single-strand tails. The xWRN-mediated end processing is remarkably similar to the end processing that has been proposed for the Escherichia coli RecQ helicase and RecJ single-strand nuclease, suggesting that this mechanism might be conserved in prokaryotes and eukaryotes.