Abstract
During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.
Highlights
In mitotically dividing cells, DNA damage such as double strand breaks (DSBs) involves potentially lethal events that must be repaired to maintain the integrity of the genome
To identify proteins phosphorylated during meiosis, diploid cells were arrested in pachytene and synchronously induced to proceed through the meiotic divisions using a conditional allele of the meiosis-specific transcription factor, NDT80 [45, 46]
Whole cell extracts were generated from cells taken at timepoints indicative of either Meiosis I or Meiosis II and the proteins digested with trypsin
Summary
DNA damage such as double strand breaks (DSBs) involves potentially lethal events that must be repaired to maintain the integrity of the genome. DSBs are programmed to occur primarily in preferred regions of the genome called “hotspots” using a highly conserved meiosis-specific, topoisomerase-like protein, Spo11 [4, 5]. These breaks are used to create crossovers (COs) between the non-sister chromatids of homologous chromosomes. Changing the bias for repair template from sister chromatids to homologs requires meiosis-specific changes to chromosome structure, the DNA damage response and recombination proteins
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