Abstract
Meiotic recombination requires the formation of programmed Spo11-dependent DNA double strand breaks (DSBs). In Saccharomyces cerevisiae, the Sae2 protein and the Mre11-Rad50-Xrs2 complex are necessary to remove the covalently attached Spo11 protein from the DNA ends, which are then resected by so far unknown nucleases. Here, we demonstrate that phosphorylation of Sae2 Ser-267 by cyclin-dependent kinase 1 (Cdk1) is required to initiate meiotic DSB resection by allowing Spo11 removal from DSB ends. This finding suggests that Cdk1 activity is required for the processing of Spo11-induced DSBs, thus providing a mechanism for coordinating DSB resection with progression through meiotic prophase. Furthermore, the helicase Sgs1 and the nucleases Exo1 and Dna2 participate in lengthening the 5'-3' resection tracts during meiosis by controlling a step subsequent to Spo11 removal.
Highlights
double strand breaks (DSBs) formation requires meiosis-specific gene products, including the evolutionary conserved topoisomerase-like enzyme Spo11, as well as the three components of the MRX complex (Mre11-Rad50-Xrs2) [2]
Sae2 Ser-267 Is Phosphorylated by cyclin-dependent kinase 1 (Cdk1) in Meiosis—Effective DSB resection in vegetative S. cerevisiae cells is promoted by Cdk1 activity during the S and G2 phases of the cell cycle [14, 15]
We exploited the fact that Cdk1-mediated control of DSB resection during mitosis relies on the phosphorylation of Sae2 Ser267 by Cdk1 [16]
Summary
Yeast Strains—Yeast strains used for this work are listed in supplemental Table S1. All the strains were SK1 derivatives that were isogenic with the NKY3000 strain (MATa/MAT␣, HO/HO, lys2/lys, ura3::hisG/ura3::hisG, leu2::hisG/leu2::hisG), kindly provided by N. Heterozygous diploid strains carrying deletions of SAE2, DMC1, EXO1, and DNA2 genes were obtained by onestep PCR disruption. The diploid strain carrying the cdc28-as allele was kindly provided by S. ApaI digestion of the integrative plasmids pML469, pML674, pML673, pML692, pML703, pML691.3, pML691.5, and pML704 was used to direct the integration of these plasmids to the SAE2 promoter region of a SK1-derivative sae2⌬ strain, giving rise to heterozygous diploid strains carrying single copies of the SAE2, sae2S267A, sae2-S267D, sae2-S134A, sae2-S134D, sae2-S267AS179A, sae2-S267A-S134A, and sae2-S267A-S134D alleles, respectively, at the SAE2 chromosomal locus. PCR one-step tagging was used to obtain strains carrying myc-tagged SPO11 and HA-tagged SAE2, sae2-S267A, or MEK1 alleles. Meiotic DSB Formation and Processing—DSB formation and repair analysis were performed at the YCR048W locus as described [28]. Secondary antibodies were purchased from Amersham Biosciences, and proteins were visualized by using an enhanced chemiluminescence system according to the manufacturer’s instructions
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