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

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

Genetics ◽

10.1093/genetics/iyab102 ◽

2021 ◽

Author(s):

Krishnaprasad G Nandanan ◽

Sagar Salim ◽

Ajith V Pankajam ◽

Miki Shinohara ◽

Gen Lin ◽

...

Keyword(s):

Binding Sites ◽

Holliday Junction ◽

Crossing Over ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Meiotic Chromosomes ◽

Genome Wide ◽

Double Holliday Junction

Abstract 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.

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Systematic Mutagenesis of the Saccharomyces cerevisiae MLH1 Gene Reveals Distinct Roles for Mlh1p in Meiotic Crossing Over and in Vegetative and Meiotic Mismatch Repair

Molecular and Cellular Biology ◽

10.1128/mcb.23.3.873-886.2003 ◽

2003 ◽

Vol 23 (3) ◽

pp. 873-886 ◽

Cited By ~ 56

Author(s):

Juan Lucas Argueso ◽

Amanda Wraith Kijas ◽

Sumeet Sarin ◽

Julie Heck ◽

Marc Waase ◽

...

Keyword(s):

Mismatch Repair ◽

Ternary Complex ◽

Genetic Recombination ◽

Crossing Over ◽

Wild Type ◽

Dna Mismatch ◽

Biochemical Assays ◽

Postmeiotic Segregation ◽

New Alleles

ABSTRACT 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.

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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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Exo1-protected DNA nicks direct crossover formation in meiosis

10.1101/2021.08.29.458102 ◽

2021 ◽

Author(s):

Michael Gioia ◽

Lisette Payero ◽

Gianno Pannafino ◽

Jun Jie Chen ◽

Sagar Salim ◽

...

Keyword(s):

Experimental Evidence ◽

Mismatch Repair ◽

Molecular Level ◽

Holliday Junction ◽

Crossing Over ◽

Structural Elements ◽

Crossover Interference ◽

Double Holliday Junction ◽

Dna Nicks ◽

Null Mutants

In most sexually reproducing organisms crossing over between chromosome homologs during meiosis is critical for the viability of haploid gametes. Most crossovers that form in meiosis in budding yeast result from the biased resolution of double Holliday Junction (dHJ) intermediates. This dHJ resolution step involves the actions Rad2/XPG family nuclease Exo1 and the Mlh1- Mlh3 mismatch repair endonuclease. At present little is known about how these factors act in meiosis at the molecular level. Here we show that Exo1 promotes meiotic crossing over by protecting DNA nicks from ligation. We found that structural elements in Exo1 required for interactions with DNA, such as bending of DNA during nick/flap recognition, are critical for its role in crossing over. Consistent with these observations, meiotic expression of the Rad2/XPG family member Rad27 partially rescued the crossover defect in exo1 null mutants, and meiotic overexpression of Cdc9 ligase specifically reduced the crossover levels of exo1 DNA binding mutants to levels approaching the exo1 null. In addition, our work identified a role for Exo1 in crossover interference that appears independent of its resection activity. Together, these studies provide experimental evidence for Exo1 protected nicks being critical for the formation of meiotic crossovers and their distribution.

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

Genetics ◽

10.1093/genetics/151.4.1245 ◽

1999 ◽

Vol 151 (4) ◽

pp. 1245-1259 ◽

Cited By ~ 4

Author(s):

Michèle Vedel ◽

Alain Nicolas

Keyword(s):

Gene Conversion ◽

Mismatch Repair ◽

Meiotic Recombination ◽

Physical Analysis ◽

Wild Type ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Meiotic Gene ◽

Allelic Differences

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 Budding Yeast Msh4 Protein Functions in Chromosome Synapsis and the Regulation of Crossover Distribution

Genetics ◽

10.1093/genetics/158.3.1013 ◽

2001 ◽

Vol 158 (3) ◽

pp. 1013-1025 ◽

Cited By ~ 7

Author(s):

Janet E Novak ◽

Petra B Ross-Macdonald ◽

G Shirleen Roeder

Keyword(s):

Mismatch Repair ◽

Budding Yeast ◽

Null Mutation ◽

Crossing Over ◽

Wild Type ◽

Crossover Interference ◽

Meiotic Chromosomes ◽

Chromosome Synapsis ◽

Protein Functions ◽

Or Gene

AbstractThe budding yeast MSH4 gene encodes a MutS homolog produced specifically in meiotic cells. Msh4 is not required for meiotic mismatch repair or gene conversion, but it is required for wild-type levels of crossing over. Here, we show that a msh4 null mutation substantially decreases crossover interference. With respect to the defect in interference and the level of crossing over, msh4 is similar to the zip1 mutant, which lacks a structural component of the synaptonemal complex (SC). Furthermore, epistasis tests indicate that msh4 and zip1 affect the same subset of meiotic crossovers. In the msh4 mutant, SC formation is delayed compared to wild type, and full synapsis is achieved in only about half of all nuclei. The simultaneous defects in synapsis and interference observed in msh4 (and also zip1 and ndj1/tam1) suggest a role for the SC in mediating interference. The Msh4 protein localizes to discrete foci on meiotic chromosomes and colocalizes with Zip2, a protein involved in the initiation of chromosome synapsis. Both Zip2 and Zip1 are required for the normal localization of Msh4 to chromosomes, raising the possibility that the zip1 and zip2 defects in crossing over are indirect, resulting from the failure to localize Msh4 properly.

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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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The Conversion Gradient at HIS4 of Saccharomyces cerevisiae. II. A Role for Mismatch Repair Directed by Biased Resolution of the Recombinational Intermediate

Genetics ◽

10.1093/genetics/153.2.573 ◽

1999 ◽

Vol 153 (2) ◽

pp. 573-583 ◽

Cited By ~ 10

Author(s):

Henriette M Foss ◽

Kenneth J Hillers ◽

Franklin W Stahl

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Synthesis ◽

Mismatch Repair ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Holliday Junction ◽

Crossing Over ◽

Gradient Type ◽

Salient Features

AbstractSalient features of recombination at ARG4 of Saccharomyces provoke a variation of the double-strand-break repair (DSBR) model that has the following features: (1) Holliday junction cutting is biased in favor of strands upon which DNA synthesis occurred during formation of the joint molecule (this bias ensures that cutting both junctions of the joint-molecule intermediate arising during DSBR usually leads to crossing over); (2) cutting only one junction gives noncrossovers; and (3) repair of mismatches that are semirefractory to mismatch repair and/or far from the DSB site is directed primarily by junction resolution. The bias in junction resolution favors restoration of 4:4 segregation when such mismatches and the directing junction are on the same side of the DSB site. Studies at HIS4 confirmed the predicted influence of the bias in junction resolution on the conversion gradient, type of mismatch repair, and frequency of aberrant 5:3 segregation, as well as the predicted relationship between mismatch repair and crossing over.

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The Arabidopsis HOP2 Gene Has a Role in Preventing illegitimate Exchanges between Nonhomologous Chromosomes

10.1101/2021.08.04.455073 ◽

2021 ◽

Author(s):

YIsell Farahani-Tafreshi ◽

Chun Wei ◽

Peilu Gan ◽

Jenya Daradur ◽

C. Daniel Riggs ◽

...

Keyword(s):

Active Role ◽

Crossing Over ◽

Wild Type ◽

Positive Role ◽

Homologous Chromosomes ◽

Chromosome Synapsis ◽

Radiation Induced ◽

Nonhomologous Chromosome ◽

Chromosome Exchanges

Meiotic homologous chromosomes pair up and undergo crossing over. In many eukaryotes both intimate pairing and crossing over require the induction of double stranded breaks (DSBs) and subsequent repair via Homologous Recombination (HR). In these organisms, two key proteins are the recombinases RAD51 and DMC1. Recombinase-modulators HOP2 and MND1 have been identified as proteins that assist RAD51 and DMC1 and are needed to promote stabilized pairing. We have probed the nature of the genetic lesions seen in hop2 mutants and looked at the role of HOP2 in the fidelity of genetic exchanges. Using γH2AX as a marker for unrepaired DSBs we found that hop2-1 and mnd1 mutants have different appearance/disappearance for DSBs than wild type, but all DSBs are repaired by mid-late pachytene. Therefore, the bridges and fragments seen from metaphase I onward are due to mis-repaired DSBs, not unrepaired ones. Studying Arabidopsis haploid meiocytes we found that wild type haploids produced the expected five univalents, but hop2-1 haploids suffered many illegitimate exchanges that were stable enough to produce bridged chromosomes during segregation. Our results suggest that HOP2 has a significant active role in preventing nonhomologous associations. We also found evidence that HOP2 plays a role in preventing illegitimate exchanges during repair of radiation-induced DSBs in rapidly dividing petal cells. Thus, HOP2 plays both a positive role in promoting homologous chromosome synapsis and a separable role in preventing nonhomologous chromosome exchanges. Possible mechanisms for this second important role are discussed.

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Ndj1, a Telomere-Associated Protein, Promotes Meiotic Recombination in Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.26.10.3683-3694.2006 ◽

2006 ◽

Vol 26 (10) ◽

pp. 3683-3694 ◽

Cited By ~ 40

Author(s):

Hsin-Yen Wu ◽

Sean M. Burgess

Keyword(s):

Meiotic Recombination ◽

Double Strand Breaks ◽

Homology Search ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Synaptonemal Complex Formation ◽

Strand Invasion ◽

Double Holliday Junction ◽

Meiosis I

ABSTRACT Dynamic telomere repositioning is a prominent feature of meiosis. Deletion of a telomere-associated protein, Ndj1, results in the failure of both attachment and clustering of telomeres at the nuclear envelope and delays several landmarks of meiosis I, such as pairing, synaptonemal complex formation, and timing of the meiosis I division. We explored the role of Ndj1 in meiotic recombination, which occurs through the formation and repair of programmed double-strand breaks. The ndj1Δ mutation allows for the formation of the first detectable strand invasion intermediate (i.e., single-end invasion) with wild-type kinetics; however, it confers a delay in the formation of the double-Holliday junction intermediate and both crossover and noncrossover products. These results challenge the widely held notion that clustering of telomeres in meiosis promotes the ability of homologous chromosomes to find one another in budding Saccharomyces cerevisiae. We propose that an Ndj1-dependent function is critical for stabilizing analogous strand invasion intermediates that exist in two separate branches of the bifurcated pathway, leading to either noncrossover or crossover formation. These findings provide a link between telomere dynamics and a distinct mechanistic step of meiotic recombination that follows the homology search.

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