Coordinated and Independent Roles for MLH Subunits in DNA Repair

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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Mnd1/Hop2 Facilitates Dmc1-Dependent Interhomolog Crossover Formation in Meiosis of Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.26.8.2913-2923.2006 ◽

2006 ◽

Vol 26 (8) ◽

pp. 2913-2923 ◽

Cited By ~ 31

Author(s):

Jill M. Henry ◽

Raymond Camahort ◽

Douglas A. Rice ◽

Laurence Florens ◽

Selene K. Swanson ◽

...

Keyword(s):

Meiotic Recombination ◽

Double Mutant ◽

Repetitive Sequences ◽

Double Strand Breaks ◽

Genomic Integrity ◽

Dna Interactions ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Meiotic Crossover ◽

Strand Invasion

ABSTRACT 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.

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CRISPR targeting of MEIOTIC-TOPOISOMERASE VIB-dCas9 to a recombination hotspot is insufficient to increase crossover frequency in Arabidopsis

10.1101/2021.02.01.429210 ◽

2021 ◽

Author(s):

Nataliya E. Yelina ◽

Sabrina Gonzalez-Jorge ◽

Dominique Hirsz ◽

Ziyi Yang ◽

Ian R. Henderson

Keyword(s):

Meiotic Recombination ◽

Crop Improvement ◽

Double Strand Breaks ◽

Crossover Frequency ◽

Dna Double Strand Breaks ◽

Crop Breeding ◽

Catalytic Subunits ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Meiotic Crossover

AbstractDuring meiosis, homologous chromosomes pair and recombine, which can result in reciprocal crossovers that increase genetic diversity. Crossovers are unevenly distributed along eukaryote chromosomes and show repression in heterochromatin and the centromeres. Within the chromosome arms crossovers are often concentrated in hotspots, which are typically in the kilobase range. The uneven distribution of crossovers along chromosomes, together with their low number per meiosis, creates a limitation during crop breeding, where recombination can be beneficial. Therefore, targeting crossovers to specific genome locations has the potential to accelerate crop improvement. In plants, meiotic crossovers are initiated by DNA double strand breaks (DSBs) that are catalysed by SPO11 complexes, which consist of two catalytic (SPO11-1 and SPO11-2) and two non-catalytic subunits (MTOPVIB). We used the model plant Arabidopsis thaliana to target a dCas9-MTOPVIB fusion protein to the 3a crossover hotspot via CRISPR. We observed that this was insufficient to significantly change meiotic crossover frequency or pattern within 3a. We discuss the implications of our findings for targeting meiotic recombination within plant genomes.

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The bakers’s yeast Msh4-Msh5 associates with double-strand break hotspots and chromosome axis during meiosis to promote crossovers

10.1101/2020.07.24.219295 ◽

2020 ◽

Author(s):

Krishnaprasad G. Nandanan ◽

Ajith V. Pankajam ◽

Sagar Salim ◽

Miki Shinohara ◽

Gen Lin ◽

...

Keyword(s):

Mismatch Repair ◽

Baker’S Yeast ◽

Double Strand Breaks ◽

Holliday Junction ◽

Baker's Yeast ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Genome Wide ◽

Chromosome Axis

ABSTRACTSegregation of homologous chromosomes during the first meiotic division requires at least one obligate crossover/exchange event between the homolog pairs. In the baker’s yeast Saccharomyces cerevisiae and mammals, the mismatch repair-related factors, Msh4-Msh5 and Mlh1-Mlh3 generate the majority of the meiotic crossovers from programmed double-strand breaks (DSBs). To understand the mechanistic role of Msh4-Msh5 in meiotic crossing over, we performed genome-wide ChIP-sequencing and cytological analysis of the Msh5 protein in cells synchronized for meiosis. We observe that Msh5 associates with DSB hotspots, chromosome axis, and centromeres. We found that the initial recruitment of Msh4-Msh5 occurs following DSB resection. A two-step Msh5 binding pattern was observed: an early weak binding at DSB hotspots followed by enhanced late binding upon the formation of double Holliday junction structures. Msh5 association with the chromosome axis is Red1 dependent, while Msh5 association with the DSB hotspots and axis is dependent on DSB formation by Spo11. Msh5 binding was enhanced at strong DSB hotspots consistent with a role for DSB frequency in promoting Msh5 binding. These data on the in vivo localization of Msh5 during meiosis have implications for how Msh4-Msh5 may work with other crossover and synapsis promoting factors to ensure Holliday junction resolution at the chromosome axis.AUTHOR SUMMARYDuring meiosis, crossovers facilitate physical linkages between homologous chromosomes that ensure their accurate segregation. Meiotic crossovers are initiated from programmed DNA double-strand breaks (DSBs). In the baker’s yeast and mammals, DSBs are repaired into crossovers primarily through a pathway involving the highly conserved mismatch repair related Msh4-Msh5 complex along with other crossover promoting factors. In vitro and physical studies suggest that the Msh4-Msh5 heterodimer facilitates meiotic crossover formation by stabilizing Holliday junctions. We investigated the genome-wide in vivo binding sites of Msh5 during meiotic progression. Msh5 was enriched at DSB hotspots, chromosome axis, and centromere sites. Our results suggest Msh5 associates with both DSB sites on the chromosomal loops and with the chromosome axis to promote crossover formation. These results on the in vivo dynamic localization of the Msh5 protein provide novel insights into how the Msh4-Msh5 complex may work with other crossover and synapsis promoting factors to facilitate crossover formation.

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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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Massive crossover elevation via combination of HEI10 and recq4a recq4b during Arabidopsis meiosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1713071115 ◽

2018 ◽

Vol 115 (10) ◽

pp. 2437-2442 ◽

Cited By ~ 38

Author(s):

Heïdi Serra ◽

Christophe Lambing ◽

Catherine H. Griffin ◽

Stephanie D. Topp ◽

Divyashree C. Nageswaran ◽

...

Keyword(s):

Meiotic Recombination ◽

Homologous Chromosome ◽

Crop Improvement ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Meiotic Dsbs ◽

Competing Pathways

During meiosis, homologous chromosomes undergo reciprocal crossovers, which generate genetic diversity and underpin classical crop improvement. Meiotic recombination initiates from DNA double-strand breaks (DSBs), which are processed into single-stranded DNA that can invade a homologous chromosome. The resulting joint molecules can ultimately be resolved as crossovers. In Arabidopsis, competing pathways balance the repair of ∼100–200 meiotic DSBs into ∼10 crossovers per meiosis, with the excess DSBs repaired as noncrossovers. To bias DSB repair toward crossovers, we simultaneously increased dosage of the procrossover E3 ligase gene HEI10 and introduced mutations in the anticrossovers helicase genes RECQ4A and RECQ4B. As HEI10 and recq4a recq4b increase interfering and noninterfering crossover pathways, respectively, they combine additively to yield a massive meiotic recombination increase. Interestingly, we also show that increased HEI10 dosage increases crossover coincidence, which indicates an effect on interference. We also show that patterns of interhomolog polymorphism and heterochromatin drive recombination increases distally towards the subtelomeres in both HEI10 and recq4a recq4b backgrounds, while the centromeres remain crossover suppressed. These results provide a genetic framework for engineering meiotic recombination landscapes in plant genomes.

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The Yeast Motor Protein, Kar3p, Is Essential for Meiosis I

The Journal of Cell Biology ◽

10.1083/jcb.139.2.459 ◽

1997 ◽

Vol 139 (2) ◽

pp. 459-467 ◽

Cited By ~ 29

Author(s):

Carol A. Bascom-Slack ◽

Dean S. Dawson

Keyword(s):

Synaptonemal Complex ◽

Meiotic Recombination ◽

Molecular Motor ◽

Motor Protein ◽

Double Strand Breaks ◽

Wild Type ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Low Levels ◽

Meiosis I

The recognition and alignment of homologous chromosomes early in meiosis is essential for their subsequent segregation at anaphase I; however, the mechanism by which this occurs is unknown. We demonstrate here that, in the absence of the molecular motor, Kar3p, meiotic cells are blocked with prophase monopolar microtubule arrays and incomplete synaptonemal complex (SC) formation. kar3 mutants exhibit very low levels of heteroallelic recombination. kar3 mutants do produce double-strand breaks that act as initiation sites for meiotic recombination in yeast, but at levels severalfold reduced from wild-type. These data are consistent with a meiotic role for Kar3p in the events that culminate in synapsis of homologues.

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SPO16 binds SHOC1 to promote homologous recombination and crossing-over in meiotic prophase I

Science Advances ◽

10.1126/sciadv.aau9780 ◽

2019 ◽

Vol 5 (1) ◽

pp. eaau9780 ◽

Cited By ~ 3

Author(s):

Qianting Zhang ◽

Shu-Yan Ji ◽

Kiran Busayavalasa ◽

Chao Yu

Keyword(s):

Homologous Recombination ◽

Meiotic Recombination ◽

Double Strand Breaks ◽

Crossing Over ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Recombination Nodules ◽

Evolutionarily Conserved

Segregation of homologous chromosomes in meiosis I is tightly regulated by their physical links, or crossovers (COs), generated from DNA double-strand breaks (DSBs) through meiotic homologous recombination. In budding yeast, three ZMM (Zip1/2/3/4, Mer3, Msh4/5) proteins, Zip2, Zip4, and Spo16, form a “ZZS” complex, functioning to promote meiotic recombination via a DSB repair pathway. Here, we identified the mammalian ortholog of Spo16, termed SPO16, which interacts with the mammalian ortholog of Zip2 (SHOC1/MZIP2), and whose functions are evolutionarily conserved to promote the formation of COs. SPO16 localizes to the recombination nodules, as SHOC1 and TEX11 do. SPO16 is required for stabilization of SHOC1 and proper localization of other ZMM proteins. The DSBs formed in SPO16-deleted meiocytes were repaired without COs formation, although synapsis is less affected. Therefore, formation of SPO16-SHOC1 complex–associated recombination intermediates is a key step facilitating meiotic recombination that produces COs from yeast to mammals.

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Mouse TEX15 is essential for DNA double-strand break repair and chromosomal synapsis during male meiosis

The Journal of Cell Biology ◽

10.1083/jcb.200709057 ◽

2008 ◽

Vol 180 (4) ◽

pp. 673-679 ◽

Cited By ~ 75

Author(s):

Fang Yang ◽

Sigrid Eckardt ◽

N. Adrian Leu ◽

K. John McLaughlin ◽

Peijing Jeremy Wang

Keyword(s):

Meiotic Recombination ◽

Strand Break ◽

Male Meiosis ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Recombination Proteins ◽

Meiotic Chromosomes ◽

Dna Double Strand Break

During meiosis, homologous chromosomes undergo synapsis and recombination. We identify TEX15 as a novel protein that is required for chromosomal synapsis and meiotic recombination. Loss of TEX15 function in mice causes early meiotic arrest in males but not in females. Specifically, TEX15-deficient spermatocytes exhibit a failure in chromosomal synapsis. In mutant spermatocytes, DNA double-strand breaks (DSBs) are formed, but localization of the recombination proteins RAD51 and DMC1 to meiotic chromosomes is severely impaired. Based on these data, we propose that TEX15 regulates the loading of DNA repair proteins onto sites of DSBs and, thus, its absence causes a failure in meiotic recombination.

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Mammalian CNTD1 is critical for meiotic crossover maturation and deselection of excess precrossover sites

The Journal of Cell Biology ◽

10.1083/jcb.201401122 ◽

2014 ◽

Vol 205 (5) ◽

pp. 633-641 ◽

Cited By ~ 45

Author(s):

J. Kim Holloway ◽

Xianfei Sun ◽

Rayka Yokoo ◽

Anne M. Villeneuve ◽

Paula E. Cohen

Keyword(s):

Chromosome Segregation ◽

Meiotic Recombination ◽

Homologous Chromosome ◽

Ring Finger ◽

Double Strand Breaks ◽

Related Protein ◽

Strand Breaks ◽

Meiotic Crossover ◽

Specific Factors ◽

Ring Finger Proteins

Meiotic crossovers (COs) are crucial for ensuring accurate homologous chromosome segregation during meiosis I. Because the double-strand breaks (DSBs) that initiate meiotic recombination greatly outnumber eventual COs, this process requires exquisite regulation to narrow down the pool of DSB intermediates that may form COs. In this paper, we identify a cyclin-related protein, CNTD1, as a critical mediator of this process. Disruption of Cntd1 results in failure to localize CO-specific factors MutLγ and HEI10 at designated CO sites and also leads to prolonged high levels of pre-CO intermediates marked by MutSγ and RNF212. These data show that maturation of COs is intimately coupled to deselection of excess pre-CO sites to yield a limited number of COs and that CNTD1 coordinates these processes by regulating the association between the RING finger proteins HEI10 and RNF212 and components of the CO machinery.

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The Zip4 protein directly couples meiotic crossover formation to synaptonemal complex assembly

Genes & Development ◽

10.1101/gad.348973.121 ◽

2021 ◽

Author(s):

Alexandra Pyatnitskaya ◽

Jessica Andreani ◽

Raphaël Guérois ◽

Arnaud De Muyt ◽

Valérie Borde

Keyword(s):

Synaptonemal Complex ◽

Central Element ◽

Underlying Mechanism ◽

Double Strand Breaks ◽

General Mechanism ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Evolutionarily Conserved ◽

Meiotic Crossover ◽

Chromosome Axis

Meiotic recombination is triggered by programmed double-strand breaks (DSBs), a subset of these being repaired as crossovers, promoted by eight evolutionarily conserved proteins, named ZMM. Crossover formation is functionally linked to synaptonemal complex (SC) assembly between homologous chromosomes, but the underlying mechanism is unknown. Here we show that Ecm11, a SC central element protein, localizes on both DSB sites and sites that attach chromatin loops to the chromosome axis, which are the starting points of SC formation, in a way that strictly requires the ZMM protein Zip4. Furthermore, Zip4 directly interacts with Ecm11, and point mutants that specifically abolish this interaction lose Ecm11 binding to chromosomes and exhibit defective SC assembly. This can be partially rescued by artificially tethering interaction-defective Ecm11 to Zip4. Mechanistically, this direct connection ensuring SC assembly from CO sites could be a way for the meiotic cell to shut down further DSB formation once enough recombination sites have been selected for crossovers, thereby preventing excess crossovers. Finally, the mammalian ortholog of Zip4, TEX11, also interacts with the SC central element TEX12, suggesting a general mechanism.

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