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Crossover invariance determined by partner choice for meiotic DNA break repair.

ABSTRACT: Crossovers between meiotic homologs are crucial for their proper segregation, and crossover number and position are carefully controlled. Crossover homeostasis in budding yeast maintains crossovers at the expense of noncrossovers when double-strand DNA break (DSB) frequency is reduced. The mechanism of maintaining constant crossover levels in other species has been unknown. Here we investigate in fission yeast a different aspect of crossover control--the near invariance of crossover frequency per kb of DNA despite large variations in DSB intensity across the genome. Crossover invariance involves the choice of sister chromatid versus homolog for DSB repair. At strong DSB hotspots, intersister repair outnumbers interhomolog repair approximately 3:1, but our genetic and physical data indicate the converse in DSB-cold regions. This unanticipated mechanism of crossover control may operate in many species and explain, for example, the large excess of DSBs over crossovers and the repair of DSBs on unpaired chromosomes in diverse species.

SUBMITTER: Hyppa RW

PROVIDER: S-EPMC2911445 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Crossover invariance determined by partner choice for meiotic DNA break repair.

Hyppa Randy W RW Smith Gerald R GR

Cell 20100701 2

Crossovers between meiotic homologs are crucial for their proper segregation, and crossover number and position are carefully controlled. Crossover homeostasis in budding yeast maintains crossovers at the expense of noncrossovers when double-strand DNA break (DSB) frequency is reduced. The mechanism of maintaining constant crossover levels in other species has been unknown. Here we investigate in fission yeast a different aspect of crossover control--the near invariance of crossover frequency pe ...[more]

PMID: 20655467

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Project description:During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.

Project description:Repair of programmed DNA double-strand breaks (DSBs) by meiotic recombination relies on the generation of flanking 3' single-stranded DNA overhangs and their interaction with a homologous double-stranded DNA template. In various common model organisms, the ubiquitous strand exchange protein Rad51 and its meiosis-specific homologue Dmc1 have been implicated in the joint promotion of DNA-strand exchange at meiotic recombination sites. However, the division of labor between these two recombinases is still a puzzle. Using RNAi and gene-disruption experiments, we have studied their roles in meiotic recombination and chromosome pairing in the ciliated protist Tetrahymena as an evolutionarily distant meiotic model. Cytological and electrophoresis-based assays for DSBs revealed that, without Rad51p, DSBs were not repaired. However, in the absence of Dmc1p, efficient Rad51p-dependent repair took place, but crossing over was suppressed. Immunostaining and protein tagging demonstrated that only Dmc1p formed strong DSB-dependent foci on meiotic chromatin, whereas the distribution of Rad51p was diffuse within nuclei. This suggests that meiotic nucleoprotein filaments consist primarily of Dmc1p. Moreover, a proximity ligation assay confirmed that little if any Rad51p forms mixed nucleoprotein filaments with Dmc1p. Dmc1p focus formation was independent of the presence of Rad51p. The absence of Dmc1p did not result in compensatory assembly of Rad51p repair foci, and even artificial DNA damage by UV failed to induce Rad51p foci in meiotic nuclei, while it did so in somatic nuclei within one and the same cell. The observed interhomologue repair deficit in dmc1Δ meiosis is consistent with a requirement for Dmc1p in promoting the homologue as the preferred recombination partner. We propose that relatively short and/or transient Rad51p nucleoprotein filaments are sufficient for intrachromosomal recombination, whereas long nucleoprotein filaments consisting primarily of Dmc1p are required for interhomolog recombination.

Dataset Information

Crossover invariance determined by partner choice for meiotic DNA break repair.

Publications

Crossover invariance determined by partner choice for meiotic DNA break repair.

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