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

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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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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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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ZCWPW1 is recruited to recombination hotspots by PRDM9, and is essential for meiotic double strand break repair

10.1101/821678 ◽

2019 ◽

Cited By ~ 5

Author(s):

Daniel Wells ◽

Emmanuelle Bitoun ◽

Daniela Moralli ◽

Gang Zhang ◽

Anjali Gupta Hinch ◽

...

Keyword(s):

Binding Sites ◽

Protein S ◽

Strand Break ◽

Double Strand Breaks ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Histone Marks ◽

Cpg Dinucleotides ◽

Recombination Hotspots ◽

Genome Wide

AbstractDuring meiosis, homologous chromosomes pair (synapse) and recombine, enabling balanced segregation and generating genetic diversity. In many vertebrates, recombination initiates with double-strand breaks (DSBs) within hotspots where PRDM9 binds, and deposits H3K4me3 and H3K36me3. However, no protein(s) recognising this unique combination of histone marks have yet been identified.We identified Zcwpw1, which possesses H3K4me3 and H3K36me3 recognition domains, as highly co-expressed with Prdm9. Here, we show that ZCWPW1 has co-evolved with PRDM9 and, in human cells, is strongly and specifically recruited to PRDM9 binding sites, with higher affinity than sites possessing H3K4me3 alone. Surprisingly, ZCWPW1 also recognizes CpG dinucleotides, including within many Alu transposons.Male Zcwpw1 homozygous knockout mice show completely normal DSB positioning, but persistent DMC1 foci at many hotspots, particularly those more strongly bound by PRDM9, severe DSB repair and synapsis defects, and downstream sterility. Our findings suggest a model where ZCWPW1 recognition of PRDM9-bound sites on either the homologous, or broken, chromosome is critical for synapsis, and hence fertility.Graphical Abstract LegendIn humans and other species, recombination is initiated by double strand breaks at sites bound by PRDM9. Upon binding, PRDM9 deposits the histone marks H3K4me3 and H3K36me, but the functional importance of these marks has remained unknown. Here, we show that PRDM9 recruits ZCWPW1, a reader of both these marks, to its binding sites genome-wide. ZCWPW1 does not help position the breaks themselves, but is essential for their downstream repair and chromosome pairing, and ultimately meiotic success and fertility in mice.

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De novo deletion mutations at recombination hotspots in mouse germlines

10.1101/2020.06.23.168138 ◽

2020 ◽

Author(s):

Agnieszka Lukaszewicz ◽

Julian Lange ◽

Scott Keeney ◽

Maria Jasin

Keyword(s):

De Novo ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Atm Kinase ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Recombination Hotspots ◽

Genome Wide ◽

Meiotic Dsbs ◽

Meiotic Recombination Hotspots

AbstractNumerous DNA double-strand breaks (DSBs) arise genome-wide during meiosis to ensure recombination between homologous chromosomes, which is required for gamete formation1,2. The ATM kinase plays a central role in controlling both the number and position of DSBs3-5, but the consequences of deregulated DSB formation have not been explored. Here we discovered that an unanticipated type of DNA deletion arises at meiotic recombination hotspots in the absence of ATM. Deletions form via joining of ends from two closely-spaced DSBs at adjacent hotspots or within a single hotspot. Deletions are also detected in normal cells, albeit at much lower frequency, revealing that the meiotic genome has a hidden potential for deletion events. Remarkably, a subset of deletions contain insertions that likely originated from DNA fragments released from hotspots on other chromosomes. Moreover, although deletions form primarily within one chromosome, joining between homologous chromosomes is also observed. This predicts in turn gross chromosome rearrangements, with evidence of damage to multiple chromatids and aborted gap repair. Thus, multiple nearby meiotic DSBs are normally suppressed by ATM to protect genomic integrity. We expect the de novo germline mutations we observe to affect human health and genome evolution.

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Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

10.1101/469635 ◽

2018 ◽

Cited By ~ 7

Author(s):

Beeke Wienert ◽

Stacia K Wyman ◽

Christopher D Richardson ◽

Charles D Yeh ◽

Pinar Akcakaya ◽

...

Keyword(s):

Gene Editing ◽

Double Strand Breaks ◽

Therapeutic Gene ◽

Strand Breaks ◽

Cellular Models ◽

Genome Wide ◽

Target Sites ◽

Single Base Resolution

AbstractGenome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach takes advantage of the recruitment of endogenous DNA repair factors for genome-wide identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides an unprecedented view of events that precede repair of the affected sites.

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

10.1101/2021.08.13.456249 ◽

2021 ◽

Author(s):

Alexandra Pyatnitskaya ◽

Jessica Andreani ◽

Raphael Guerois ◽

Arnaud De Muyt ◽

Valerie 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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Multilayered mechanisms ensure that short chromosomes recombine in meiosis

10.1101/406892 ◽

2018 ◽

Cited By ~ 2

Author(s):

Hajime Murakami ◽

Isabel Lam ◽

Jacquelyn Song ◽

Megan van Overbeek ◽

Scott Keeney

Keyword(s):

Saccharomyces Cerevisiae ◽

Selection Pressure ◽

Homologous Chromosome ◽

Replication Timing ◽

Double Strand Breaks ◽

Strand Breaks ◽

Homologous Chromosomes ◽

Chromosome Axis

To segregate accurately during meiosis, homologous chromosomes in most species must recombine. Very small chromosomes would risk missegregation if recombination were randomly distributed, so the double-strand breaks (DSBs) that initiate recombination are not haphazard. How this nonrandomness is controlled is not under-stood. Here we demonstrate that Saccharomyces cerevisiae integrates multiple, temporally distinct pathways to regulate chromosomal binding of pro-DSB factors Rec114 and Mer2, thereby controlling duration of a DSB-competent state. Homologous chromosome engagement regulates Rec114/Mer2 dissociation late in prophase, whereas replication timing and proximity to centromeres or telomeres influence timing and amount of Rec114/Mer2 accumulation early. A distinct early mechanism boosts Rec114/Mer2 binding quickly to high levels specifically on the shortest chromosomes, dependent on chromosome axis proteins and subject to selection pressure to maintain hyperrecombinogenic properties of these chromosomes. Thus, an organism’s karyotype and its attendant risk of meiotic missegregation influence the shape and evolution of its recombination landscape.

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ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis

10.1101/2019.12.18.881409 ◽

2019 ◽

Cited By ~ 2

Author(s):

Jacob Paiano ◽

Wei Wu ◽

Shintaro Yamada ◽

Nicholas Sciascia ◽

Elsa Callen ◽

...

Keyword(s):

Meiotic Recombination ◽

Homologous Chromosome ◽

Direct Detection ◽

Double Strand Breaks ◽

Sequence Specificity ◽

Dna Sequence Specificity ◽

Strand Breaks ◽

Genome Wide ◽

Meiotic Dsbs

AbstractMeiotic recombination is initiated by genome-wide SPO11-induced double-strand breaks (DSBs) that are processed by MRE11-mediated release of SPO11. The DSB is then resected and loaded with DMC1/RAD51 filaments that invade homologous chromosome templates. In most mammals, DSB locations (“hotspots”) are determined by the DNA sequence specificity of PRDM9. Here, we demonstrate the first direct detection of meiotic DSBs and resection in vertebrates by performing END-seq on mouse spermatocytes using low sample input. We find that DMC1 limits both the minimum and maximum lengths of resected DNA, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles in meiotic resection. Through enzymatic modifications to the END-seq protocol that mimic the in vivo processing of SPO11, we identify a novel meiotic recombination intermediate (“SPO11-RI”) present at all hotspots. The SPO11-bound intermediate is dependent on PRDM9 and caps the 3’ resected end during engagement with the homologous template. We propose that SPO11-RI is generated because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm−/− spermatocytes, SPO11-RI is reduced while unresected DNA-bound SPO11 accumulate because of defective MRE11 initiation. Thus in addition to their global roles in governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.

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