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PLoS Genetics Mar 2024During meiosis, genetic recombination is initiated by the formation of many DNA double-strand breaks (DSBs) catalysed by the evolutionarily conserved topoisomerase-like...
During meiosis, genetic recombination is initiated by the formation of many DNA double-strand breaks (DSBs) catalysed by the evolutionarily conserved topoisomerase-like enzyme, Spo11, in preferred genomic sites known as hotspots. DSB formation activates the Tel1/ATM DNA damage responsive (DDR) kinase, locally inhibiting Spo11 activity in adjacent hotspots via a process known as DSB interference. Intriguingly, in S. cerevisiae, over short genomic distances (<15 kb), Spo11 activity displays characteristics of concerted activity or clustering, wherein the frequency of DSB formation in adjacent hotspots is greater than expected by chance. We have proposed that clustering is caused by a limited number of sub-chromosomal domains becoming primed for DSB formation. Here, we provide evidence that DSB clustering is abolished when meiotic prophase timing is extended via deletion of the NDT80 transcription factor. We propose that extension of meiotic prophase enables most cells, and therefore most chromosomal domains within them, to reach an equilibrium state of similar Spo11-DSB potential, reducing the impact that priming has on estimates of coincident DSB formation. Consistent with this view, when Tel1 is absent but Ndt80 is present and thus cells are able to rapidly exit meiotic prophase, genome-wide maps of Spo11-DSB formation are skewed towards pericentromeric regions and regions that load pro-DSB factors early-revealing regions of preferential priming-but this effect is abolished when NDT80 is deleted. Our work highlights how the stochastic nature of Spo11-DSB formation in individual cells within the limited temporal window of meiotic prophase can cause localised DSB clustering-a phenomenon that is exacerbated in tel1Δ cells due to the dual roles that Tel1 has in DSB interference and meiotic prophase checkpoint control.
Topics: DNA; DNA Breaks, Double-Stranded; DNA-Binding Proteins; Endodeoxyribonucleases; Intracellular Signaling Peptides and Proteins; Meiosis; Prophase; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 38427688
DOI: 10.1371/journal.pgen.1011140 -
Nucleic Acids Research May 2024Accurate chromosome segregation during meiosis requires the establishment of at least one crossover (CO) between each pair of homologous chromosomes. CO formation...
Accurate chromosome segregation during meiosis requires the establishment of at least one crossover (CO) between each pair of homologous chromosomes. CO formation depends on a group of conserved pro-CO proteins, which colocalize at CO-designated sites during late meiotic prophase I. However, it remains unclear whether these pro-CO proteins form a functional complex and how they promote meiotic CO formation in vivo. Here, we show that COSA-1, a key component required for CO formation, interacts with other pro-CO factors, MSH-5 and ZHP-3, via its N-terminal disordered region. Point mutations that impair these interactions do not affect CO designation, but they strongly hinder the accumulation of COSA-1 at CO-designated sites and result in defective CO formation. These defects can be partially bypassed by artificially tethering an interaction-compromised COSA-1 derivate to ZHP-3. Furthermore, we revealed that the accumulation of COSA-1 into distinct foci is required to assemble functional 'recombination nodules'. These prevent early CO-designated recombination intermediates from being dismantled by the RTEL-1 helicase and protect late recombination intermediates, such as Holliday junctions, until they are resolved by CO-specific resolvases. Altogether, our findings provide insight into COSA-1 mediated pro-CO complex assembly and its contribution to CO formation.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Chromosome Segregation; Crossing Over, Genetic; DNA-Binding Proteins; Meiosis
PubMed: 38412290
DOI: 10.1093/nar/gkae130 -
Nucleic Acids Research May 2024Meiotic recombination is of central importance for the proper segregation of homologous chromosomes, but also for creating genetic diversity. It is initiated by the...
Meiotic recombination is of central importance for the proper segregation of homologous chromosomes, but also for creating genetic diversity. It is initiated by the formation of double-strand breaks (DSBs) in DNA catalysed by evolutionarily conserved Spo11, together with additional protein partners. Difficulties in purifying the Spo11 protein have limited the characterization of its biochemical properties and of its interactions with other DSB proteins. In this study, we have purified fragments of Spo11 and show for the first time that Spo11 can physically interact with Mre11 and modulates its DNA binding, bridging, and nuclease activities. The interaction of Mre11 with Spo11 requires its far C-terminal region, which is in line with the severe meiotic phenotypes of various mre11 mutations located at the C-terminus. Moreover, calibrated ChIP for Mre11 shows that Spo11 promotes Mre11 recruitment to chromatin, independent of DSB formation. A mutant deficient in Spo11 interaction severely reduces the association of Mre11 with meiotic chromatin. Consistent with the reduction of Mre11 foci in this mutant, it strongly impedes DSB formation, leading to spore death. Our data provide evidence that physical interaction between Spo11 and Mre11, together with end-bridging, promote normal recruitment of Mre11 to hotspots and DSB formation.
Topics: Chromatin; DNA Breaks, Double-Stranded; DNA-Binding Proteins; Endodeoxyribonucleases; Exodeoxyribonucleases; Meiosis; Mutation; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 38407383
DOI: 10.1093/nar/gkae111 -
BioRxiv : the Preprint Server For... Feb 2024According to Mendel's second law, chromosomes segregate randomly in meiosis. Nonrandom segregation is primarily known for cases of selfish meiotic drive in females, in...
According to Mendel's second law, chromosomes segregate randomly in meiosis. Nonrandom segregation is primarily known for cases of selfish meiotic drive in females, in which particular alleles bias their own transmission into the oocyte. Here, we report a rare example of unselfish meiotic drive for crossover inheritance in the clonal raider ant, . This species produces diploid offspring parthenogenetically via fusion of two haploid nuclei from the same meiosis. This process should cause rapid genotypic degeneration due to loss of heterozygosity, which results if crossover recombination is followed by random (Mendelian) segregation of chromosomes. However, by comparing whole genomes of mothers and daughters, we show that loss of heterozygosity is exceedingly rare, raising the possibility that crossovers are infrequent or absent in meiosis. Using a combination of cytology and whole genome sequencing, we show that crossover recombination is, in fact, common, but that loss of heterozygosity is avoided because crossover products are faithfully co-inherited. This results from a programmed violation of Mendel's law of segregation, such that crossover products segregate together rather than randomly. This discovery highlights an extreme example of cellular "memory" of crossovers, which could be a common yet cryptic feature of chromosomal segregation.
PubMed: 38405725
DOI: 10.1101/2024.02.09.579553 -
Nature Communications Feb 2024Drugs targeting the DNA damage response (DDR) are widely used in cancer therapy, but resistance to these drugs remains a major clinical challenge. Here, we show that...
Drugs targeting the DNA damage response (DDR) are widely used in cancer therapy, but resistance to these drugs remains a major clinical challenge. Here, we show that SYCP2, a meiotic protein in the synaptonemal complex, is aberrantly and commonly expressed in breast and ovarian cancers and associated with broad resistance to DDR drugs. Mechanistically, SYCP2 enhances the repair of DNA double-strand breaks (DSBs) through transcription-coupled homologous recombination (TC-HR). SYCP2 promotes R-loop formation at DSBs and facilitates RAD51 recruitment independently of BRCA1. SYCP2 loss impairs RAD51 localization, reduces TC-HR, and renders tumors sensitive to PARP and topoisomerase I (TOP1) inhibitors. Furthermore, our studies of two clinical cohorts find that SYCP2 overexpression correlates with breast cancer resistance to antibody-conjugated TOP1 inhibitor and ovarian cancer resistance to platinum treatment. Collectively, our data suggest that SYCP2 confers cancer cell resistance to DNA-damaging agents by stimulating R-loop-mediated DSB repair, offering opportunities to improve DDR therapy.
Topics: R-Loop Structures; DNA Repair; DNA Breaks, Double-Stranded; Homologous Recombination; BRCA1 Protein; DNA; Rad51 Recombinase; Recombinational DNA Repair
PubMed: 38383600
DOI: 10.1038/s41467-024-45693-2 -
PLoS Genetics Feb 2024Meiotic recombination between homologous chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs). Approximately 10% of these DSBs...
Meiotic recombination between homologous chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs). Approximately 10% of these DSBs result in crossovers (COs), sites of physical DNA exchange between homologs that are critical to correct chromosome segregation. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers, the latter representing the defining marks of CO sites. The regulation of CO number and position is poorly understood, but undoubtedly requires the coordinated action of multiple repair pathways. In a previous report, we found gene-trap disruption of the DNA helicase, FANCJ (BRIP1/BACH1), elicited elevated numbers of MLH1 foci and chiasmata. In somatic cells, FANCJ interacts with numerous DNA repair proteins including MLH1, and we hypothesized that FANCJ functions with MLH1 to regulate the major CO pathway. To further elucidate the meiotic function of FANCJ, we produced three new Fancj mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, truncation of the N-terminal Helicase domain, and a C-terminal dual-tagged allele. We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, none of our Fancj mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 in meiosis. Instead, FANCJ co-localizes with BRCA1 and TOPBP1, forming discrete foci along the chromosome cores beginning in early meiotic prophase I and densely localized to unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Fancj mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data indicate a role for FANCJ in early DSB repair, but they rule out a role for FANCJ in MLH1-mediated CO events.
Topics: Animals; Male; Mice; Alleles; DNA Helicases; DNA Repair; Meiosis; Meiotic Prophase I
PubMed: 38377115
DOI: 10.1371/journal.pgen.1011175 -
BioRxiv : the Preprint Server For... Feb 2024A bipolar spindle composed of microtubules and many associated proteins functions to segregate chromosomes during cell division in all eukaryotes, yet spindle size and...
A bipolar spindle composed of microtubules and many associated proteins functions to segregate chromosomes during cell division in all eukaryotes, yet spindle size and architecture varies dramatically across different species and cell types. Targeting protein for Xklp2 (TPX2) is one candidate factor for modulating spindle microtubule organization through its roles in branching microtubule nucleation, activation of the mitotic kinase Aurora A, and association with the kinesin-5 (Eg5) motor. Here we identify a conserved nuclear localization sequence (NLS) motif, KKLK in TPX2, which regulates astral microtubule formation and spindle pole morphology in egg extracts. Addition of recombinant TPX2 with this sequence mutated to AALA dramatically increased spontaneous formation of microtubule asters and recruitment of phosphorylated Aurora A, pericentrin, and Eg5 to meiotic spindle poles. We propose that TPX2 is a linchpin spindle assembly factor whose regulation contributes to the recruitment and activation of multiple microtubule polymerizing and organizing proteins, generating distinct spindle architectures.
PubMed: 38370704
DOI: 10.1101/2024.02.10.579770 -
Science Advances Feb 2024The synaptonemal complex (SC) is a zipper-like protein assembly that links homologous chromosomes to regulate recombination and segregation during meiosis. The SC has...
The synaptonemal complex (SC) is a zipper-like protein assembly that links homologous chromosomes to regulate recombination and segregation during meiosis. The SC has been notoriously refractory to in vitro reconstitution, thus leaving its molecular organization largely unknown. Here, we report a moonlighting function of two paralogous S-phase kinase-associated protein 1 (Skp1)-related proteins (SKR-1 and SKR-2), well-known adaptors of the Skp1-Cul1-F-box (SCF) ubiquitin ligase, as the key missing components of the SC in . SKR proteins repurpose their SCF-forming interfaces to dimerize and interact with meiosis-specific SC proteins, thereby driving synapsis independent of SCF activity. SKR-1 enables the formation of the long-sought-after soluble complex with previously identified SC proteins in vitro, which we propose it to represent a complete SC building block. Our findings demonstrate how a conserved cell cycle regulator has been co-opted to interact with rapidly evolving meiotic proteins to construct the SC and provide a foundation for understanding its structure and assembly mechanisms.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Cycle Proteins; S-Phase Kinase-Associated Proteins; Synaptonemal Complex
PubMed: 38354250
DOI: 10.1126/sciadv.adl4876 -
BioRxiv : the Preprint Server For... Apr 2024The classical phenomenon of crossover interference is a one-dimensional spatial patterning process that produces evenly spaced crossovers during meiosis. Quantitative...
The classical phenomenon of crossover interference is a one-dimensional spatial patterning process that produces evenly spaced crossovers during meiosis. Quantitative analysis of diagnostic molecules along budding yeast chromosomes reveals that this process also sets up a second, interdigitated pattern of related but longer periodicity, in a "two-tiered" patterning process. The second tier corresponds to a previously mysterious minority set of crossovers. Thus, in toto, the two tiers account for all detected crossover events. Both tiers of patterning set up spatially clustered assemblies of three types of molecules ("triads") representing the three major components of meiotic chromosomes (crossover recombination complexes and chromosome axis and synaptonemal complex components), and give focal and domainal signals, respectively. Roles are suggested. All observed effects are economically and synthetically explained if crossover patterning is mediated by mechanical forces along prophase chromosomes. Intensity levels of domainal triad components are further modulated, dynamically, by the conserved protein remodeler Pch2/TRIP13.
PubMed: 38352537
DOI: 10.1101/2024.01.28.577645 -
BioRxiv : the Preprint Server For... Feb 2024Very little is known about the process of meiosis in the apicomplexan parasite despite the essentiality of sex in its life cycle. Most cell lines only support asexual...
Very little is known about the process of meiosis in the apicomplexan parasite despite the essentiality of sex in its life cycle. Most cell lines only support asexual growth of (), but stem cell derived intestinal epithelial cells grown under air-liquid interface (ALI) conditions support the sexual cycle. To examine chromosomal dynamics during meiosis in , we generated two transgenic lines of parasites that were fluorescently tagged with mCherry or GFP on chromosomes 1 or 5, respectively. Infection of ALI cultures or mice with mCherry and GFP parasites produced "yellow" oocysts generated by cross-fertilization. Outcrossed oocysts from the F generation were purified and used to infect HCT-8 cultures, and phenotypes of the progeny were observed by microscopy. All possible phenotypes predicted by independent segregation were represented equally (~25%) in the population, indicating that chromosomes exhibit a Mendelian inheritance pattern. Unexpectedly, the most common pattern observed from the outgrowth of single oocysts included all possible parental and recombinant phenotypes derived from a single meiotic event, suggesting a high rate of crossover. To estimate the frequency of crossover, additional loci on chromosomes 1 and 5 were tagged and used to monitor intrachromosomal crosses in mice. Both chromosomes showed a high frequency of crossover compared to other apicomplexans with map distances (i.e., 1% recombination) of 3-12 kb. Overall, a high recombination rate may explain many unique characteristics observed in spp. such as high rates of speciation, wide variation in host range, and rapid evolution of host-specific virulence factors.
PubMed: 38352509
DOI: 10.1101/2024.02.02.578536