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The Mnd1 protein forms a complex with hop2 to promote homologous chromosome pairing and meiotic double-strand break repair.

ABSTRACT: The hop2 mutant of Saccharomyces cerevisiae arrests in meiosis with extensive synaptonemal complex (SC) formation between nonhomologous chromosomes. A screen for multicopy suppressors of a hop2-ts allele identified the MND1 gene. The mnd1-null mutant arrests in meiotic prophase, with most double-strand breaks (DSBs) unrepaired. A low level of mature recombinants is produced, and the Rad51 protein accumulates at numerous foci along chromosomes. SC formation is incomplete, and homolog pairing is severely reduced. The Mnd1 protein localizes to chromatin throughout meiotic prophase, and this localization requires Hop2. Unlike recombination enzymes such as Rad51, Mnd1 localizes to chromosomes even in mutants that fail to initiate meiotic recombination. The Hop2 and Mnd1 proteins coimmunoprecipitate from meiotic cell extracts. These results suggest that Hop2 and Mnd1 work as a complex to promote meiotic chromosome pairing and DSB repair. The identification of Hop2 and Mnd1 homologs in other organisms suggests that the function of this complex is conserved among eukaryotes.

SUBMITTER: Tsubouchi H

PROVIDER: S-EPMC133769 | biostudies-literature | 2002 May

REPOSITORIES: biostudies-literature

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The Mnd1 protein forms a complex with hop2 to promote homologous chromosome pairing and meiotic double-strand break repair.

Tsubouchi Hideo H Roeder G Shirleen GS

Molecular and cellular biology 20020501 9

The hop2 mutant of Saccharomyces cerevisiae arrests in meiosis with extensive synaptonemal complex (SC) formation between nonhomologous chromosomes. A screen for multicopy suppressors of a hop2-ts allele identified the MND1 gene. The mnd1-null mutant arrests in meiotic prophase, with most double-strand breaks (DSBs) unrepaired. A low level of mature recombinants is produced, and the Rad51 protein accumulates at numerous foci along chromosomes. SC formation is incomplete, and homolog pairing is s ...[more]

PMID: 11940665

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Project description:Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Consequently, cells in all domains of life have evolved mechanisms to repair DSBs, including through homologous recombination. Although recombination has been well characterized, the spatial organization of this process in living cells remains poorly understood. Here, we introduced site-specific DSBs in Caulobacter crescentus, and then used time-lapse microscopy to visualize the homology search, DSB repair, and the resegregation of chromosomal DNA. Even loci tethered to opposite cell poles can efficiently release, pair to enable recombination-based repair, and then resegregate to their original locations. Resegregation occurs independent of DNA replication and without disrupting global chromosome organization. Origin-proximal regions are resegregated by the same machinery, ParABS, used to segregate undamaged chromosomes following DNA replication. In contrast, origin-distal regions efficiently resegregate after a DSB independent of ParABS, and likely without dedicated segregation proteins. Instead, we propose that a physical, spring-like force drives the resegregation of origin-distal loci after DSB repair. Caulobacter cells were depleted of DnaA for 1.5 h before synchronization. Swarmer cells were then released into DnaA depleting conditions (without IPTG) and double-strand breaks were induced for 1 h by the addition of 500 ?M vanillate. For control sample, no vanillate was added. Formadehyde (Sigma) was then added to the final concentration of 1%. Formadehyde crosslinks protein-DNA and DNA-DNA together, thereby capturing the structure of the chromosome at the time of fixation. Fixation was performed at the cell density of OD600 = 0.2. The crosslinking reactions were allowed to proceed for 30 minutes at 25 °C before quenching with 2.5 M glycine at a final concentration of 0.125 M. Fixed cells were then pelleted by centrifugation and subsequently washed twice with 1x M2 buffer (6.1 mM Na2HPO4, 3.9 mM KH2PO4, 9.3 mM NH4Cl, 0.5 mM MgSO4, 10 ?M FeSO4, 0.5 mM CaCl2) before resuspending in 1x TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA) to a final concentration of 107 cells per µl. Resuspended cells were then divided into 25 µl aliquots and stored at -80 °C for no more than 2 weeks. Each Hi-C experiment was performed using two of the 25 µL aliquots. Chromosome conformation capture with next-generation seqeuncing (Hi-C) was carried out exactly as described previously (Le et al., 2013 PMID: 24158908)

Dataset Information

The Mnd1 protein forms a complex with hop2 to promote homologous chromosome pairing and meiotic double-strand break repair.

Publications

The Mnd1 protein forms a complex with hop2 to promote homologous chromosome pairing and meiotic double-strand break repair.

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