CYS3, a Hotspot of Meiotic Recombination in Saccharomyces cerevisiae: Effects of Heterozygosity and Mismatch Repair Functions on Gene Conversion and Recombination Intermediates

Abstract We have examined meiotic recombination at the CYS3 locus. Genetic analysis indicates that CYS3 is a hotspot of meiotic gene conversion, with a putative 5′–3′ polarity gradient of conversion frequencies. This gradient is relieved in the presence of msh2 and pms1 mutations, indicating an involvement of mismatch repair functions in meiotic recombination. To investigate the role of mismatch repair proteins in meiotic recombination, we performed a physical analysis of meiotic DNA in wild-type and msh2 pms1 strains in the presence or absence of allelic differences at CYS3. Neither the mutations in CYS3 nor the absence of mismatch repair functions affects the frequency and distribution of nearby recombination-initiating DNA double-strand breaks (DSBs). Processing of DSBs is also similar in msh2 pms1 and wild-type strains. We conclude that mismatch repair functions do not control the distribution of meiotic gene conversion events at the initiating steps. In the MSH2 PMS1 background, strains heteroallelic for frameshift mutations in CYS3 exhibit a frequency of gene conversion greater than that observed for either marker alone. Physical analysis revealed no modification in the formation of DSBs, suggesting that this marker effect results from subsequent processing events that are not yet understood.

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The Role of the Mismatch Repair Machinery in Regulating Mitotic and Meiotic Recombination Between Diverged Sequences in Yeast

Genetics ◽

10.1093/genetics/151.4.1299 ◽

1999 ◽

Vol 151 (4) ◽

pp. 1299-1313 ◽

Cited By ~ 13

Author(s):

Wenliang Chen ◽

Sue Jinks-Robertson

Keyword(s):

Mismatch Repair ◽

Meiotic Recombination ◽

Type Strain ◽

Wild Type Strain ◽

Sequence Divergence ◽

Rate Data ◽

Wild Type ◽

Recombination Rates ◽

Sequence Identity

Abstract Nonidentical recombination substrates recombine less efficiently than do identical substrates in yeast, and much of this inhibition can be attributed to action of the mismatch repair (MMR) machinery. In this study an intron-based inverted repeat assay system has been used to directly compare the rates of mitotic and meiotic recombination between pairs of 350-bp substrates varying from 82% to 100% in sequence identity. The recombination rate data indicate that sequence divergence impacts mitotic and meiotic recombination similarly, although subtle differences are evident. In addition to assessing recombination rates as a function of sequence divergence, the endpoints of mitotic and meiotic recombination events involving 94%-identical substrates were determined by DNA sequencing. The endpoint analysis indicates that the extent of meiotic heteroduplex DNA formed in a MMR-defective strain is 65% longer than that formed in a wild-type strain. These data are consistent with a model in which the MMR machinery interferes with the formation and/or extension of heteroduplex intermediates during recombination.

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Control of meiotic recombination in Arabidopsis: role of the MutL and MutS homologues

Biochemical Society Transactions ◽

10.1042/bst0340542 ◽

2006 ◽

Vol 34 (4) ◽

pp. 542-544 ◽

Cited By ~ 23

Author(s):

F.C.H. Franklin ◽

J.D. Higgins ◽

E. Sanchez-Moran ◽

S.J. Armstrong ◽

K.E. Osman ◽

...

Keyword(s):

Mismatch Repair ◽

Meiotic Recombination ◽

Holliday Junction ◽

Crossing Over ◽

Wild Type ◽

Pathway Model ◽

Junction Formation ◽

Double Holliday Junction ◽

Dependent Pathway

Immunocytochemistry reveals that the Arabidopsis mismatch repair proteins AtMSH4, AtMLH3 and AtMLH1 are expressed during prophase I of meiosis. Expression of AtMSH4 precedes AtMLH3 and AtMLH1 which co-localize as foci during pachytene. Co-localization between AtMSH4 and AtMLH3 occurs, but appears transient. AtMLH3 foci are not detected in an Atmsh4 mutant. However, localization of AtMSH4 is unaffected in Atmlh3, suggesting that recombination may proceed to dHj (double Holliday junction) formation. Mean chiasma frequency in Atmsh4 is reduced to 1.55 compared with 9.86 in wild-type. In contrast with wild-type, the distribution of residual crossovers in Atmsh4 closely fits a Poisson distribution. This is consistent with a two-pathway model for meiotic crossing-over whereby most crossovers occur via an AtMSH4-dependent pathway that is subject to interference, with the remaining crossovers arising via an interference-independent pathway. Loss of AtMLH3 results in an approx. 60% reduction in crossovers. Results suggest that dHj resolution can occur, but in contrast with wild-type where most or all dHjs are directed to form crossovers, the outcome is biased in favour of a non-crossover outcome. The results are compatible with a model whereby the MutL complex maintains or imposes a dHj conformation that ensures crossover formation.

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Repair of Double-Strand Breaks by Homologous Recombination in Mismatch Repair-Defective Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.21.8.2671-2682.2001 ◽

2001 ◽

Vol 21 (8) ◽

pp. 2671-2682 ◽

Cited By ~ 115

Author(s):

Beth Elliott ◽

Maria Jasin

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Mismatch Repair ◽

Mammalian Cells ◽

Es Cells ◽

Embryonic Stem ◽

Double Strand Breaks ◽

Wild Type ◽

Strand Breaks ◽

Mutant Cells

ABSTRACT Chromosomal double-strand breaks (DSBs) stimulate homologous recombination by several orders of magnitude in mammalian cells, including murine embryonic stem (ES) cells, but the efficiency of recombination decreases as the heterology between the repair substrates increases (B. Elliott, C. Richardson, J. Winderbaum, J. A. Nickoloff, and M. Jasin, Mol. Cell. Biol. 18:93–101, 1998). We have now examined homologous recombination in mismatch repair (MMR)-defective ES cells to investigate both the frequency of recombination and the outcome of events. Using cells with a targeted mutation in the msh2 gene, we found that the barrier to recombination between diverged substrates is relaxed for both gene targeting and intrachromosomal recombination. Thus, substrates with 1.5% divergence are 10-fold more likely to undergo DSB-promoted recombination in Msh2 −/− cells than in wild-type cells. Although mutant cells can repair DSBs efficiently, examination of gene conversion tracts in recombinants demonstrates that they cannot efficiently correct mismatched heteroduplex DNA (hDNA) that is formed adjacent to the DSB. As a result, >20-fold more of the recombinants derived from mutant cells have uncorrected tracts compared with recombinants from wild-type cells. The results indicate that gene conversion repair of DSBs in mammalian cells frequently involves mismatch correction of hDNA rather than double-strand gap formation. In cells with MMR defects, therefore, aberrant recombinational repair may be an additional mechanism that contributes to genomic instability and possibly tumorigenesis.

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A Role for Synaptonemal Complex in Meiotic Mismatch Repair

Genetics ◽

10.1093/genetics/iyab230 ◽

2021 ◽

Author(s):

Karen Voelkel-Meiman ◽

Ashwini Oke ◽

Arden Feil ◽

Alexander Shames ◽

Jennifer Fung ◽

...

Keyword(s):

Synaptonemal Complex ◽

Gene Conversion ◽

Mismatch Repair ◽

Meiotic Recombination ◽

High Throughput Sequencing ◽

Central Element ◽

Similar Degree ◽

Meiotic Segregation ◽

Wild Type ◽

A Genome

Abstract A large subset of meiotic recombination intermediates form within the physical context of synaptonemal complex (SC), but the functional relationship between SC structure and homologous recombination remains obscure. Our prior analysis of strains deficient for SC central element proteins demonstrated that tripartite SC is dispensable for interhomolog recombination in S. cerevisiae. Here we report that while dispensable for recombination per se, SC proteins promote efficient mismatch repair at interhomolog recombination sites. Failure to repair mismatches within heteroduplex-containing meiotic recombination intermediates leads to genotypically sectored colonies (post meiotic segregation events). We discovered increased post-meiotic segregation at THR1 in cells lacking Ecm11 or Gmc2, or in the SC-deficient but recombination-proficient zip1[Δ21-163] mutant. High-throughput sequencing of octad meiotic products furthermore revealed a genome-wide increase in recombination events with unrepaired mismatches in ecm11 mutants relative to wild type. Meiotic cells missing Ecm11 display longer gene conversion tracts, but tract length alone does not account for the higher frequency of unrepaired mismatches. Interestingly, the per-nucleotide mismatch frequency is elevated in ecm11 when analyzing all gene conversion tracts, but is similar between wild type and ecm11 if considering only those events with unrepaired mismatches. Thus, in both wild type and ecm11 strains a subset of recombination events is susceptible to a similar degree of inefficient mismatch repair, but in ecm11 mutants a larger fraction of events fall into this inefficient repair category. Finally, we observe elevated post-meiotic segregation at THR1 in mutants with a dual deficiency in MutSγ crossover recombination and SC assembly, but not in the mlh3 mutant, which lacks MutSγ crossovers but has abundant SC. We propose that SC structure promotes efficient mismatch repair of joint molecule recombination intermediates, and that absence of SC is the molecular basis for elevated post-meiotic segregation in both MutSγ crossover-proficient (ecm11, gmc2) and MutSγ crossover-deficient (msh4, zip3) strains.

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Functional Interactions Between SPO11 and REC102 During Initiation of Meiotic Recombination in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/160.1.111 ◽

2002 ◽

Vol 160 (1) ◽

pp. 111-122

Author(s):

Kehkooi Kee ◽

Scott Keeney

Keyword(s):

Saccharomyces Cerevisiae ◽

Meiotic Recombination ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Whole Cells ◽

Meiotic Gene ◽

Meiotic Chromosomes ◽

Cell Extracts ◽

Cold Sensitive

Abstract In Saccharomyces cerevisiae, formation of the DNA double-strand breaks (DSBs) that initiate meiotic recombination requires the products of at least 10 genes. Spo11p is thought to be the catalytic subunit of the DNA cleaving activity, but the roles of the other proteins, and the interactions among them, are not well understood. This study demonstrates genetic and physical interactions between the products of SPO11 and another early meiotic gene required for DSB formation, REC102. We found that epitope-tagged versions of SPO11 and REC102 that by themselves were capable of supporting normal or nearly normal levels of meiotic recombination conferred a severe synthetic cold-sensitive phenotype when combined in the same cells. DSB formation, meiotic gene conversion, and spore viability were drastically reduced in the doubly tagged strain at a nonpermissive temperature. This conditional defect could be partially rescued by expression of untagged SPO11, but not by expression of untagged REC102, indicating that tagged REC102 is fully dominant for this synthetic phenotype. Both tagged and wild-type Spo11p co-immunoprecipitated with tagged Rec102p from meiotic cell extracts, indicating that these proteins are present in a common complex in vivo. Tagged Rec102p localized to the nucleus in whole cells and to chromatin on spread meiotic chromosomes. Our results are consistent with the idea that a multiprotein complex that includes Spo11p and Rec102p promotes meiotic DSB formation.

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Saccharomyces cerevisiae Checkpoint Genes MEC1, RAD17 and RAD24 Are Required for Normal Meiotic Recombination Partner Choice

Genetics ◽

10.1093/genetics/153.2.607 ◽

1999 ◽

Vol 153 (2) ◽

pp. 607-620 ◽

Cited By ~ 4

Author(s):

Jeremy M Grushcow ◽

Teresa M Holzen ◽

Ken J Park ◽

Ted Weinert ◽

Michael Lichten ◽

...

Keyword(s):

Meiotic Recombination ◽

Mitotic Checkpoint ◽

Partner Choice ◽

Wild Type ◽

Spore Viability ◽

Checkpoint Signaling ◽

Ectopic Recombination ◽

Meiotic Progression ◽

Checkpoint Genes

Abstract Checkpoint gene function prevents meiotic progression when recombination is blocked by mutations in the recA homologue DMC1. Bypass of dmc1 arrest by mutation of the DNA damage checkpoint genes MEC1, RAD17, or RAD24 results in a dramatic loss of spore viability, suggesting that these genes play an important role in monitoring the progression of recombination. We show here that the role of mitotic checkpoint genes in meiosis is not limited to maintaining arrest in abnormal meioses; mec1-1, rad24, and rad17 single mutants have additional meiotic defects. All three mutants display Zip1 polycomplexes in two- to threefold more nuclei than observed in wild-type controls, suggesting that synapsis may be aberrant. Additionally, all three mutants exhibit elevated levels of ectopic recombination in a novel physical assay. rad17 mutants also alter the fraction of recombination events that are accompanied by an exchange of flanking markers. Crossovers are associated with up to 90% of recombination events for one pair of alleles in rad17, as compared with 65% in wild type. Meiotic progression is not required to allow ectopic recombination in rad17 mutants, as it still occurs at elevated levels in ndt80 mutants that arrest in prophase regardless of checkpoint signaling. These observations support the suggestion that MEC1, RAD17, and RAD24, in addition to their proposed monitoring function, act to promote normal meiotic recombination.

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Mechanisms of Recombination between Diverged Sequences in Wild-Type and BLM-Deficient Mouse and Human Cells

Molecular and Cellular Biology ◽

10.1128/mcb.01553-09 ◽

2010 ◽

Vol 30 (8) ◽

pp. 1887-1897 ◽

Cited By ~ 40

Author(s):

Jeannine R. LaRocque ◽

Maria Jasin

Keyword(s):

Gene Conversion ◽

Mammalian Cells ◽

Sequence Divergence ◽

Dna Lesions ◽

Human Cells ◽

Deficient Mouse ◽

Wild Type ◽

Strand Breaks ◽

Major Mechanism ◽

Homeologous Recombination

ABSTRACT Double-strand breaks (DSBs) are particularly deleterious DNA lesions for which cells have developed multiple mechanisms of repair. One major mechanism of DSB repair in mammalian cells is homologous recombination (HR), whereby a homologous donor sequence is used as a template for repair. For this reason, HR repair of DSBs is also being exploited for gene modification in possible therapeutic approaches. HR is sensitive to sequence divergence, such that the cell has developed ways to suppress recombination between diverged (“homeologous”) sequences. In this report, we have examined several aspects of HR between homeologous sequences in mouse and human cells. We found that gene conversion tracts are similar for mouse and human cells and are generally ≤100 bp, even in Msh2 − / − cells which fail to suppress homeologous recombination. Gene conversion tracts are mostly unidirectional, with no observed mutations. Additionally, no alterations were observed in the donor sequences. While both mouse and human cells suppress homeologous recombination, the suppression is substantially less in the transformed human cells, despite similarities in the gene conversion tracts. BLM-deficient mouse and human cells suppress homeologous recombination to a similar extent as wild-type cells, unlike Sgs1-deficient Saccharomyces cerevisiae.

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Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.00156-17 ◽

2017 ◽

Vol 37 (24) ◽

Cited By ~ 8

Author(s):

Sucheta Arora ◽

Rajashree A. Deshpande ◽

Martin Budd ◽

Judy Campbell ◽

America Revere ◽

...

Keyword(s):

Nuclease Activity ◽

Double Strand Breaks ◽

Endonuclease Activity ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Content Type ◽

Strand Breaks ◽

Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

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Repair Kinetics of Genomic Interstrand DNA Cross-Links: Evidence for DNA Double-Strand Break-Dependent Activation of the Fanconi Anemia/BRCA Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.24.1.123-134.2004 ◽

2004 ◽

Vol 24 (1) ◽

pp. 123-134 ◽

Cited By ~ 170

Author(s):

Andreas Rothfuss ◽

Markus Grompe

Keyword(s):

Fanconi Anemia ◽

Strand Break ◽

S Phase ◽

Dna Double Strand Breaks ◽

Subsequent Formation ◽

Strand Breaks ◽

Dna Double Strand Break ◽

Cross Links ◽

Kinetics Of

ABSTRACT The detailed mechanisms of DNA interstrand cross-link (ICL) repair and the involvement of the Fanconi anemia (FA)/BRCA pathway in this process are not known. Present models suggest that recognition and repair of ICL in human cells occur primarily during the S phase. Here we provide evidence for a refined model in which ICLs are recognized and are rapidly incised by ERCC1/XPF independent of DNA replication. However, the incised ICLs are then processed further and DNA double-strand breaks (DSB) form exclusively in the S phase. FA cells are fully proficient in the sensing and incision of ICL as well as in the subsequent formation of DSB, suggesting a role of the FA/BRCA pathway downstream in ICL repair. In fact, activation of FANCD2 occurs slowly after ICL treatment and correlates with the appearance of DSB in the S phase. In contrast, activation is rapid after ionizing radiation, indicating that the FA/BRCA pathway is specifically activated upon DSB formation. Furthermore, the formation of FANCD2 foci is restricted to a subpopulation of cells, which can be labeled by bromodeoxyuridine incorporation. We therefore conclude that the FA/BRCA pathway, while being dispensable for the early events in ICL repair, is activated in S-phase cells after DSB have formed.

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Epigenetic activation of meiotic recombination in Arabidopsis centromeres via loss of H3K9me2 and non-CG DNA methylation

10.1101/160929 ◽

2017 ◽

Cited By ~ 2

Author(s):

Charles J. Underwood ◽

Kyuha Choi ◽

Christophe Lambing ◽

Xiaohui Zhao ◽

Heïdi Serra ◽

...

Keyword(s):

Dna Methylation ◽

Meiotic Recombination ◽

Double Strand Breaks ◽

Pericentromeric Heterochromatin ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Histone 3 ◽

Differential Control

AbstractEukaryotic centromeres contain the kinetochore, which connects chromosomes to the spindle allowing segregation. During meiosis centromeres are suppressed for crossovers, as recombination in these regions can cause chromosome mis-segregation. Plant centromeres are surrounded by repetitive, transposon-dense heterochromatin that is epigenetically silenced by histone 3 lysine 9 dimethylation (H3K9me2), and DNA methylation in CG and non-CG sequence contexts. Here we show that disruption of Arabidopsis H3K9me2 and non-CG DNA methylation pathways increases meiotic DNA double strand breaks (DSBs) within centromeres, whereas crossovers increase within pericentromeric heterochromatin. Increased pericentromeric crossovers in H3K9me2/non-CG mutants occurs in both inbred and hybrid backgrounds, and involves the interfering crossover repair pathway. Epigenetic activation of recombination may also account for the curious tendency of maize transposon Ds to disrupt CHROMOMETHYLASE3 when launched from proximal loci. Thus H3K9me2 and non-CG DNA methylation exert differential control of meiotic DSB and crossover formation in centromeric and pericentromeric heterochromatin.

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