Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2–MSH3 or MSH2–MSH6) or crossing over (MSH4–MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4–MSH5. The second complex involves MLH3 together with MSH2–MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2−/− males, but not females, providing an explanation for the sexual dimorphism seen in Pms2−/− mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2–MSH3 and is decreased in Msh2−/− and Msh3−/− mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.

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Distribution of repetitive DNAs and the hybrid origin of the red vizcacha rat (Octodontidae)

Genome ◽

10.1139/g11-084 ◽

2012 ◽

Vol 55 (2) ◽

pp. 105-117 ◽

Cited By ~ 12

Author(s):

E.Y. Suárez-Villota ◽

R.A. Vargas ◽

C.L. Marchant ◽

J.E. Torres ◽

N. Köhler ◽

...

Keyword(s):

Dna Sequences ◽

Repetitive Sequences ◽

Diploid Species ◽

Hybrid Origin ◽

Comparative Genomic ◽

Telomeric Dna ◽

Meiotic Chromosomes ◽

Yellow Orange ◽

Orange Fluorescence

Great genome size (GS) variations described in desert-specialist octodontid rodents include diploid species ( Octomys mimax and Octodontomys gliroides ) and putative tetraploid species ( Tympanoctomys barrerae and Pipanacoctomys aureus ). Because of its high DNA content, elevated chromosome number, and gigas effect, the genome of T. barrerae is claimed to have resulted from tetraploidy. Alternatively, the origin of its GS has been attributed to the accumulation of repetitive sequences. To better characterize the extent and origin of these repetitive DNA, self-genomic in situ hybridization (self-GISH), whole-comparative genomic hybridization (W-CGH), and conventional GISH were conducted in mitotic and meiotic chromosomes. Self-GISH on T. barrerae mitotic plates together with comparative self-GISH (using its closest relatives) discriminate a pericentromeric and a telomeric DNA fraction. As most of the repetitive sequences are pericentromeric, it seems that the large GS of T. barrerae is not due to highly repeated sequences accumulated along chromosomes arms. W-CGH using red-labeled P. aureus DNA and green-labeled O. mimax DNA simultaneously on chromosomes of T. barrerae revealed a yellow–orange fluorescence over a repetitive fraction of the karyotype. However, distinctive red-only fluorescent signals were also detected at some centromeres and telomeres, indicating closer homology with the DNA sequences of P. aureus. Conventional GISH using an excess of blocking DNA from either P. aureus or O. mimax labeled only a fraction of the T. barrerae genome, indicating its double genome composition. These data point to a hybrid nature of the T. barrerae karyotype, suggesting a hybridization event in the origin of this species.

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mlh3 separation of function and endonuclease defective mutants display an unexpected effect on meiotic recombination outcomes

10.1101/108498 ◽

2017 ◽

Cited By ~ 2

Author(s):

Najla Al-Sweel ◽

Vandana Raghavan ◽

Abhishek Dutta ◽

V. P. Ajith ◽

Luigi Di Vietro ◽

...

Keyword(s):

Dna Replication ◽

Mismatch Repair ◽

Protein Interactions ◽

Meiotic Recombination ◽

Meiotic Division ◽

Dna Mismatch Repair ◽

Crossing Over ◽

Baker's Yeast ◽

Protein Protein Interactions ◽

Dna Mismatch

AbstractMlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in baker’s yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each separation of function class were purified and characterized. Both Mlh1-mlh3-32 (MMR+, crossover-) and Mlh1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for two mlh3 mutants with opposite separation of function phenotypes, and an endonuclease defective mutant. Unexpectedly, all three showed increases in the number of non-crossover events that were not observed in mlh3Δ. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3’s enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.Author SummaryDuring meiosis, diploid germ cells that become eggs or sperm undergo a single round of DNA replication followed by two consecutive chromosomal divisions. The segregation of chromosomes at the first meiotic division is dependent in most organisms on at least one genetic exchange, or crossover event, between chromosome homologs. Homologs that do not receive a crossover frequently undergo non-disjunction at the first meiotic division, yielding gametes that lack chromosomes or contain additional copies. Such events have been linked to human disease and infertility. Recent studies suggest that the Mlh1-Mlh3 complex is an endonuclease that resolves recombination intermediates into crossovers. Interestingly, this complex also acts as a matchmaker in DNA mismatch repair (MMR) to remove DNA replication errors. How does one complex act in two different processes? We investigated this question by performing a mutational analysis of the baker’s yeast Mlh3 protein. Five mutations were identified that disrupted MMR but not crossing over, and one mutation disrupted crossing over while maintaining MMR. Using a combination of biochemical and genetic analyses to further characterize these mutants we illustrate the importance of protein-protein interactions for Mlh1-Mlh3’s activity. Importantly, we illustrate how defective meiotic components can alter the outcome of meiotic recombination events. They also provide new insights in our understanding of the basis of infertility syndromes.

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The Tn3 beta-lactamase gene acts as a hotspot for meiotic recombination in yeast.

Genetics ◽

10.1093/genetics/127.1.39 ◽

1991 ◽

Vol 127 (1) ◽

pp. 39-51 ◽

Cited By ~ 2

Author(s):

A Stapleton ◽

T D Petes

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Sequence ◽

Dna Sequences ◽

Meiotic Recombination ◽

Genetic Distances ◽

Crossing Over ◽

Beta Lactamase ◽

Yeast Saccharomyces Cerevisiae ◽

Lactamase Gene ◽

Bacterial Transposon

Abstract Although genetic distances are often assumed to be proportional to physical distances, chromosomal regions with unusually high (hotspots) or low (coldspots) levels of meiotic recombination have been described in a number of genetic systems. In general, the DNA sequences responsible for these effects have not been determined. We report that the 5' region of the beta-lactamase (ampR) gene of the bacterial transposon Tn3 is a hotspot for meiotic recombination when inserted into the chromosomes of the yeast Saccharomyces cerevisiae. When these sequences are homozygous, both crossing over and gene conversion are locally stimulated. The 5' end of the beta-lactamase gene is about 100-fold "hotter" for crossovers than an average yeast DNA sequence.

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Crossing Over During Caenorhabditis elegans Meiosis Requires a Conserved MutS-Based Pathway That Is Partially Dispensable in Budding Yeast

Genetics ◽

10.1093/genetics/153.3.1271 ◽

1999 ◽

Vol 153 (3) ◽

pp. 1271-1283 ◽

Cited By ~ 4

Author(s):

Jonathan Zalevsky ◽

Amy J MacQueen ◽

Joseph B Duffy ◽

Kenneth J Kemphues ◽

Anne M Villeneuve

Keyword(s):

Caenorhabditis Elegans ◽

Gene Loss ◽

Temperature Shift ◽

Crossing Over ◽

Direct Role ◽

Homologous Chromosomes ◽

Dna Mismatch ◽

Normal Number ◽

Differential Stability ◽

Mmr Proteins

Abstract Formation of crossovers between homologous chromosomes during Caenorhabditis elegans meiosis requires the him-14 gene. Loss of him-14 function severely reduces crossing over, resulting in lack of chiasmata between homologs and consequent missegregation. Cytological analysis showing that homologs are paired and aligned in him-14 pachytene nuclei, together with temperature-shift experiments showing that him-14 functions during the pachytene stage, indicate that him-14 is not needed to establish pairing or synapsis and likely has a more direct role in crossover formation. him-14 encodes a germline-specific member of the MutS family of DNA mismatch repair (MMR) proteins. him-14 has no apparent role in MMR, but like its Saccharomyces cerevisiae ortholog MSH4, has a specialized role in promoting crossing over during meiosis. Despite this conservation, worms and yeast differ significantly in their reliance on this pathway: whereas worms use this pathway to generate most, if not all, crossovers, yeast still form 30–50% of their normal number of crossovers when this pathway is absent. This differential reliance may reflect differential stability of crossover-competent recombination intermediates, or alternatively, the presence of two different pathways for crossover formation in yeast, only one of which predominates during nematode meiosis. We discuss a model in which HIM-14 promotes crossing over by interfering with Holliday junction branch migration.

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Factors enforcing the species boundary between the human pathogens Cryptococcus neoformans and Cryptococcus deneoformans

10.1101/2020.05.21.108084 ◽

2020 ◽

Cited By ~ 1

Author(s):

Shelby J. Priest ◽

Marco A. Coelho ◽

Verónica Mixão ◽

Shelly Clancey ◽

Yitong Xu ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Mismatch Repair ◽

Dna Sequences ◽

Meiotic Recombination ◽

Genetic Recombination ◽

Species Boundaries ◽

Repair System ◽

Hybrid Progeny ◽

Dna Mismatch ◽

Species Boundary

AbstractHybridization has resulted in the origin and variation in extant species, and hybrids continue to arise despite pre- and post-zygotic barriers that limit their formation and evolutionary success. One important system that maintains species boundaries in prokaryotes and eukaryotes is the mismatch repair pathway, which blocks recombination between divergent DNA sequences. Previous studies illuminated the role of the mismatch repair component Msh2 in blocking genetic recombination between divergent DNA during meiosis. Loss of Msh2 results in increased interspecific genetic recombination in bacterial and yeast models, and increased viability of progeny derived from yeast hybrid crosses. Hybrid isolates of two pathogenic fungal Cryptococcus species, Cryptococcus neoformans and Cryptococcus deneoformans, are isolated regularly from both clinical and environmental sources. In the present study, we sought to determine if loss of Msh2 would relax the species boundary between C. neoformans and C. deneoformans. We found that crosses between these two species in which both parents lack Msh2 produced hybrid progeny with increased viability and high levels of aneuploidy. Whole-genome sequencing revealed few instances of recombination among hybrid progeny and did not identify increased levels of recombination in progeny derived from parents lacking Msh2. Several hybrid progeny produced structures associated with sexual reproduction when incubated alone on nutrient-rich medium in light, a novel phenotype in Cryptococcus. These findings represent a unique, unexpected case where rendering the mismatch repair system defective did not result in increased meiotic recombination across a species boundary. This suggests that alternative pathways or other mismatch repair components limit meiotic recombination between homeologous DNA and enforce species boundaries in the basidiomycete Cryptococcus species.Author summarySeveral mechanisms enforce species boundaries by either preventing the formation of hybrids, known as pre-zygotic barriers, or preventing the viability and fecundity of hybrids, known as post-zygotic barriers. Despite these barriers, interspecific hybrids form at an appreciable frequency, such as hybrid isolates of the human fungal pathogenic species, Cryptococcus neoformans and Cryptococcus deneoformans, which are regularly isolated from both clinical and environmental sources. C. neoformans x C. deneoformans hybrids are typically highly aneuploid, sterile, and display phenotypes intermediate to those of either parent, although self-fertile isolates and transgressive phenotypes have been observed. One important mechanism known to enforce species boundaries or lead to incipient speciation is the DNA mismatch repair system, which blocks recombination between divergent DNA sequences during meiosis. The aim of this study was to determine if genetically deleting the DNA mismatch repair component Msh2 would relax the species boundary between C. neoformans and C. deneoformans. Progeny derived from C. neoformans x C. deneoformans crosses in which both parental strains lacked Msh2 had higher viability, and unlike previous studies in Saccharomyces, these Cryptococcus hybrid progeny had higher levels of aneuploidy and no observable increase in meiotic recombination at the whole-genome level.

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Recombination and the evolution of satellite DNA

Genetics Research ◽

10.1017/s0016672300023089 ◽

1986 ◽

Vol 47 (3) ◽

pp. 167-174 ◽

Cited By ~ 35

Author(s):

Wolfgang Stephan

Keyword(s):

Natural Selection ◽

Genetic Drift ◽

Dna Sequences ◽

Meiotic Recombination ◽

Satellite Dna ◽

Cellular Level ◽

Crossing Over ◽

Zero Crossing ◽

Copy Numbers ◽

Molecular Population Genetics

SummaryIn eukaryotic chromosomes, large blocks of satellite DNA are associated with regions of reduced meiotic recombination. No function of highly repeated, tandemly arranged DNA sequences has been identified so far at the cellular level, though the structural properties of satellite DNA are relatively well known. In studying the joint action of meiotic recombination, genetic drift and natural selection on the copy number of a family of highly repeated DNA (HRDNA), this paper looks at the structure–function debate for satellite DNA from the standpoint of molecular population genetics. It is shown that (i) HRDNA accumulates most probably in regions of near zero crossing over (heterochromatin), and that (ii), due to random genetic drift the effect of unequal crossover on copy numbers is stronger, the smaller the population size. As a consequence, highly repeated sequences are likely to persist longest (over evolutionary times) in small populations. The results are based on a fairly general class of models of unequal crossing over and natural selection which have been treated both analytically and by computer simulation.

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Coordinated and Independent Roles for MLH Subunits in DNA Repair

Cells ◽

10.3390/cells10040948 ◽

2021 ◽

Vol 10 (4) ◽

pp. 948

Author(s):

Gianno Pannafino ◽

Eric Alani

Keyword(s):

Meiotic Recombination ◽

Baker’S Yeast ◽

Double Strand Breaks ◽

Baker's Yeast ◽

Genomic Integrity ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Dna Mismatch ◽

Meiotic Crossover ◽

Mmr Proteins

The MutL family of DNA mismatch repair proteins (MMR) acts to maintain genomic integrity in somatic and meiotic cells. In baker’s yeast, the MutL homolog (MLH) MMR proteins form three heterodimeric complexes, MLH1-PMS1, MLH1-MLH2, and MLH1-MLH3. The recent discovery of human PMS2 (homolog of baker’s yeast PMS1) and MLH3 acting independently of human MLH1 in the repair of somatic double-strand breaks questions the assumption that MLH1 is an obligate subunit for MLH function. Here we provide a summary of the canonical roles for MLH factors in DNA genomic maintenance and in meiotic crossover. We then present the phenotypes of cells lacking specific MLH subunits, particularly in meiotic recombination, and based on this analysis, propose a model for an independent early role for MLH3 in meiosis to promote the accurate segregation of homologous chromosomes in the meiosis I division.

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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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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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INDUCTION OF INTRACHROMOSOMAL RECOMBINATION IN YEAST BY INHIBITION OF THYMIDYLATE BIOSYNTHESIS

Genetics ◽

10.1093/genetics/114.2.375 ◽

1986 ◽

Vol 114 (2) ◽

pp. 375-392

Author(s):

B A Kunz ◽

G R Taylor ◽

R H Haynes

Keyword(s):

Gene Conversion ◽

Dna Sequences ◽

Dna Hybridization ◽

Crossing Over ◽

Repeated Dna ◽

Yeast Saccharomyces Cerevisiae ◽

Haploid Strain ◽

Reciprocal Exchange ◽

Coli Plasmid ◽

Antifolate Drugs

ABSTRACT The biosynthesis of thymidylate in the yeast Saccharomyces cerevisiae can be inhibited by antifolate drugs. We have found that antifolate treatment enhances the formation of leucine prototrophs in a haploid strain of yeast carrying, on the same chromosome, two different mutant leu2 alleles separated by Escherichia coli plasmid sequences. That this effect is a consequence of thymine nucleotide depletion was verified by the finding that provision of exogenous thymidylate eliminates the increased production of Leu+ colonies. DNA hybridization analysis revealed that recombination, including reciprocal exchange, gene conversion and unequal sister-chromatid crossing over, between the duplicated genes gave rise to the induced Leu+ segregants. Although gene conversion unaccompanied by crossing over was responsible for the major fraction of leucine prototrophs, events involving reciprocal exchange exhibited the largest increase in frequency. These data show that recombination is induced between directly repeated DNA sequences under conditions of thymine nucleotide depletion. In addition, the results of this and previous studies are consistent with the possibility that inhibition of thymidylate biosynthesis in yeast may create a metabolic condition that provokes all forms of mitotic recombination.

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