Processing of Meiotic DNA Double Strand Breaks Requires Cyclin-dependent Kinase and Multiple Nucleases

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.During the first meiotic division, homologous maternal and paternal chromosomes are segregated. In most organisms, homologs must be physically connected to ensure their proper segregation (1). By virtue of cohesion between sister chromatids, the exchange of chromosome arms through chiasmata formation provides the physical connections between homologous chromosomes. Chiasmata are generated by recombination events, which are initiated by the formation of selfinflicted DNA double strand breaks (DSBs).3 DSB 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). In particular, a Spo11 dimer coordinately breaks both DNA strands, creating a DSB with covalent linkages between the 5Ј DNA ends and the catalytic tyrosine residue of each Spo11 monomer (3). Then, Spo11 must be removed by endonucleolytic cleavage to allow further DSB end processing by 5Ј-3Ј resection that is required to initiate homologous recombination (4). This event is promoted by the Sae2 protein and the MRX complex, which are required to catalyze the endonucleolytic removal of Spo11-linked oligonucleotides (3,5,6). In fact, budding yeast sae2⌬ cells and rad50s separation-of-function mutants allow DSB formation but are totally defective in Spo11 removal from DSB ends (3,5,(7)(8)(9). Similarly, mre11 alleles impairing Mre11 nuclease activity allow Spo11-induced DSB formation, but not Spo11 removal (10 -12), suggesting that the latter may take place by Mre11-catalyzed endonucleolytic cleavage and that Sae2 participates in this process. As recently shown, also Sae2 exhibits an endonuclease activity (13), suggesting that this protein, possibly in cooperation with MRX, may allow Spo11 removal by mediating an endonucleolytic cleavage close to the DNA end.Because DSBs are highly hazardous for genome stability, commitment to DSB resection and meiotic progression must be tightly regulated to ensure proper DSB repair. In vegetative Saccharomyces cerevisiae cells, DSB resection is promoted by the activity of the cyclin-dependent protein kinase Cdk1 (Cdc28/Clb) during...

End Resection at Double-Strand Breaks: Mechanism and Regulation

“…DSB formation and processing are highly coordinated events during meiosis, and the intermediates with Spo11 attached to ends, or products of MRX-Sae2 processing are not observed in wild-type cells (Zakharyevich et al 2010). STR-Dna2 does not contribute to resection in meiosis, except in the absence of the Dmc1 recombinase (Manfrini et al 2010;Zakharyevich et al 2010). Loss of Exo1 nuclease activity does not significantly impair meiotic recombination, indicating that the short ssDNA tails generated by MRX and Sae2 are sufficient for homologous pairing (Zakharyevich et al 2010).…”

The Meiotic Checkpoint Network: Step-by-Step through Meiotic Prophase

“…Given that a number of nucleases are involved in the resection process (Mimitou and Symington 2009;Zakharyevich et al 2010;Garcia et al 2011;Schaetzlein et al 2013), an appealing model is that the MCN ensures appropriate resection rates by activating some nucleases, while (temporarily) inhibiting others (Segurado and Diffley 2008;Manfrini et al 2010;Luo et al 2013;Souquet et al 2013). In S. cerevisiae, resection by BLM Sgs1 /DNA2, in particular, is likely only activated late in meiosis (Manfrini et al 2010;Zakharyevich et al 2010). …”

“…Resection is initiated by MRE11-dependent endonucleolytic incisions near DSBs, followed by bidirectional resection that requires both MRN and EXO1 (Zakharyevich et al 2010;Garcia et al 2011 and the 9-1-1 complex are also required to restrain hyperresection (Shinohara et al 2003;Gray et al 2013;Clerici et al 2014). Given that a number of nucleases are involved in the resection process (Mimitou and Symington 2009;Zakharyevich et al 2010;Garcia et al 2011;Schaetzlein et al 2013), an appealing model is that the MCN ensures appropriate resection rates by activating some nucleases, while (temporarily) inhibiting others (Segurado and Diffley 2008;Manfrini et al 2010;Luo et al 2013;Souquet et al 2013). In S. cerevisiae, resection by BLM Sgs1 /DNA2, in particular, is likely only activated late in meiosis (Manfrini et al 2010;Zakharyevich et al 2010).…”

The Recombinases DMC1 and RAD51 Are Functionally and Spatially Separated during Meiosis in Arabidopsis

“…Work mostly performed in Saccharomyces cerevisiae and Schizosaccharomyces pombe demonstrated that the SPO11 proteins are subsequently released together with a short DNA oligonucleotide by an endonucleolytic cleavage adjacent to the break site, mediated by the Mre11/Rad50/Xrs2-Nbs1 (MRX/N) complex in conjunction with Sae2/Com1 (Neale et al, 2005;Hartung et al, 2007;Uanschou et al, 2007;Milman et al, 2009). The DNA break sites therefore have, as demonstrated for S. cerevisiae and S. pombe, protruding 39 termini, which are further extended by resection of the 59 termini depending on the activities of the MRX/N complex and Exo1 (Cromie and Smith, 2008;Mimitou and Symington, 2008;Farah et al, 2009;Manfrini et al, 2010;Zakharyevich et al, 2010;Garcia et al, 2011). The extensive single-stranded DNA (ssDNA) overhangs are bound by the heterotrimeric replication protein A (RPA) complex with high affinity, a prerequisite for the loading of the strand-exchange proteins Rad51 and Dmc1 (reviewed in Fanning et al, 2006;Broderick et al, 2010).…”