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

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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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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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 Mus81/Mms4 Endonuclease Acts Independently of Double-Holliday Junction Resolution to Promote a Distinct Subset of Crossovers During Meiosis in Budding Yeast

Genetics ◽

10.1093/genetics/164.1.81 ◽

2003 ◽

Vol 164 (1) ◽

pp. 81-94 ◽

Cited By ~ 21

Author(s):

Teresa de los Santos ◽

Neil Hunter ◽

Cindy Lee ◽

Brittany Larkin ◽

Josef Loidl ◽

...

Keyword(s):

Budding Yeast ◽

Indirect Evidence ◽

Class Ii ◽

Suppressive Effect ◽

Class I ◽

Holliday Junction ◽

Crossing Over ◽

Crossover Interference ◽

Distinct Subset ◽

Double Holliday Junction

Abstract Current models for meiotic recombination require that crossovers derive from the resolution of a double-Holliday junction (dHJ) intermediate. In prokaryotes, enzymes responsible for HJ resolution are well characterized but the identification of a eukaryotic nuclear HJ resolvase has been elusive. Indirect evidence suggests that MUS81 from humans and fission yeast encodes a HJ resolvase. We provide three lines of evidence that Mus81/Mms4 is not the major meiotic HJ resolvase in S. cerevisiae: (1) MUS81/MMS4 is required to form only a distinct subset of crossovers; (2) rather than accumulating, dHJ intermediates are reduced in an mms4 mutant; and (3) expression of a bacterial HJ resolvase has no suppressive effect on mus81 meiotic phenotypes. Our analysis also reveals the existence of two distinct classes of crossovers in budding yeast. Class I is dependent upon MSH4/MSH5 and exhibits crossover interference, while class II is dependent upon MUS81/MMS4 and exhibits no interference. mms4 specifically reduces crossing over on small chromosomes, which are known to undergo less interference. The correlation between recombination rate and degree of interference to chromosome size may therefore be achieved by modulating the balance between class I/class II crossovers.

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Reduced Mismatch Repair of Heteroduplexes Reveals “Non”-interfering Crossing Over in Wild-Type Saccharomyces cerevisiae

Genetics ◽

10.1534/genetics.106.067603 ◽

2008 ◽

Vol 178 (3) ◽

pp. 1251-1269 ◽

Cited By ~ 25

Author(s):

Tony J. Getz ◽

Stephen A. Banse ◽

Lisa S. Young ◽

Allison V. Banse ◽

Johanna Swanson ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Mismatch Repair ◽

Crossing Over ◽

Wild Type

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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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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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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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Analysis of Conditional Mutations in the Saccharomyces cerevisiae MLH1 Gene in Mismatch Repair and in Meiotic Crossing Over

Genetics ◽

10.1093/genetics/160.3.909 ◽

2002 ◽

Vol 160 (3) ◽

pp. 909-921 ◽

Cited By ~ 1

Author(s):

Juan Lucas Argueso ◽

Daniel Smith ◽

James Yi ◽

Marc Waase ◽

Sumeet Sarin ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Family ◽

Mismatch Repair ◽

Mutational Analysis ◽

Mutation Rates ◽

Temperature Sensitive ◽

Crossing Over ◽

Wild Type ◽

Genetic Studies ◽

Conditional Mutations

Abstract In mismatch repair (MMR), members of the MLH gene family have been proposed to act as key molecular matchmakers to coordinate mismatch recognition with downstream repair functions that result in mispair excision. Two members of this gene family, MLH1 and MLH3, have also been implicated in meiotic crossing over. These diverse roles suggest that a mutational analysis of MLH genes could provide reagents required to identify interactions between gene products and to test whether the different roles ascribed to a subset of these genes can be separated. In this report we show that in Saccharomyces cerevisiae the mlh1Δ mutation confers inviability in pol3-01 strain backgrounds that are defective in the Polδ proofreading exonuclease activity. This phenotype was exploited to identify four mlh1 alleles that each confer a temperature-sensitive phenotype for viability in pol3-01 strains. In three different mutator assays, strains bearing conditional mlh1 alleles displayed wild-type or nearly wild-type mutation rates at 26°. At 35°, these strains exhibited mutation rates that approached those observed in mlh1Δ mutants. The mutator phenotype exhibited in mlh1-I296S strains was partially suppressed at 35° by EXO1 overexpression. The mlh1-F228S and -I296S mutations conferred a separation-of-function phenotype in meiosis; both mlh1-F228S and -I296S strains displayed strong defects in meiotic mismatch repair but showed nearly wild-type levels of crossing over, suggesting that the conditional mutations differentially affected MLH1 functions. These genetic studies suggest that the conditional mlh1 mutations can be used to separate the MMR and meiotic crossing-over functions of MLH1 and to identify interactions between MLH1 and downstream repair components.

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SUMO is a pervasive regulator of meiosis

eLife ◽

10.7554/elife.57720 ◽

2021 ◽

Vol 10 ◽

Author(s):

Nikhil R Bhagwat ◽

Shannon N Owens ◽

Masaru Ito ◽

Jay V Boinapalli ◽

Philip Poa ◽

...

Keyword(s):

Meiotic Prophase ◽

Protein Modification ◽

Budding Yeast ◽

Meiotic Chromosome ◽

S Phase ◽

Chromosome Organization ◽

Crossing Over ◽

Chromosome Synapsis ◽

Point Analysis ◽

Consensus Motifs

Protein modification by SUMO helps orchestrate the elaborate events of meiosis to faithfully produce haploid gametes. To date, only a handful of meiotic SUMO targets have been identified. Here we delineate a multidimensional SUMO-modified meiotic proteome in budding yeast, identifying 2747 conjugation sites in 775 targets, and defining their relative levels and dynamics. Modified sites cluster in disordered regions and only a minority match consensus motifs. Target identities and modification dynamics imply that SUMOylation regulates all levels of chromosome organization and each step of meiotic prophase I. Execution-point analysis confirms these inferences, revealing functions for SUMO in S-phase, the initiation of recombination, chromosome synapsis and crossing over. K15-linked SUMO chains become prominent as chromosomes synapse and recombine, consistent with roles in these processes. SUMO also modifies ubiquitin, forming hybrid oligomers with potential to modulate ubiquitin signaling. We conclude that SUMO plays diverse and unanticipated roles in regulating meiotic chromosome metabolism.

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mei-P22Encodes a Chromosome-Associated Protein Required for the Initiation of Meiotic Recombination inDrosophila melanogaster

Genetics ◽

10.1093/genetics/162.1.245 ◽

2002 ◽

Vol 162 (1) ◽

pp. 245-258 ◽

Cited By ~ 10

Author(s):

Hao Liu ◽

Janet K Jang ◽

Naohiro Kato ◽

Kim S McKim

Keyword(s):

Meiotic Recombination ◽

Meiotic Prophase ◽

Direct Evidence ◽

Double Strand Breaks ◽

Crossing Over ◽

Wild Type ◽

Direct Role ◽

Strand Breaks ◽

Meiotic Chromosomes ◽

X Irradiation

AbstractDouble-strand breaks (DSB) initiate meiotic recombination in a variety of organisms. Here we present genetic evidence that the mei-P22 gene is required for the induction of DSBs during meiotic prophase in Drosophila females. Strong mei-P22 mutations eliminate meiotic crossing over and suppress the sterility of DSB repair-defective mutants. Interestingly, crossing over in mei-P22 mutants can be restored to almost 50% of wild-type by X irradiation. In addition, an antibody-based assay was used to demonstrate that DSBs are not formed in mei-P22 mutants. This array of phenotypes is identical to that of mei-W68 mutants; mei-W68 encodes the Drosophila Spo11 homolog that is proposed to be an enzyme required for DSB formation. Consistent with a direct role in DSB formation, mei-P22 encodes a basic 35.7-kD protein, which, when examined by immunofluorescence, localizes to foci on meiotic chromosomes. MEI-P22 foci appear transiently in early meiotic prophase, which is when meiotic recombination is believed to initiate. By using an antibody to C(3)G as a marker for synaptonemal complex (SC) formation, we observed that SC is present before MEI-P22 associates with the chromosomes, thus providing direct evidence that the development of SC precedes the initiation of meiotic recombination. Similarly, we found that MEI-P22 foci did not appear in a c(3)G mutant in which SC does not form, suggesting that DSB formation is dependent on SC formation in Drosophila. We propose that MEI-P22 interacts with meiosis-specific chromosome proteins to facilitate DSB creation by MEI-W68.

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