Abstract
DNA end resection plays a critical function in DNA double-strand break repair pathway choice. Resected DNA ends are refractory to end-joining mechanisms and are instead channeled to homology-directed repair. Using biochemical, genetic, and imaging methods, we show that phosphorylation of Saccharomyces cerevisiae Sae2 controls its capacity to promote the Mre11-Rad50-Xrs2 (MRX) nuclease to initiate resection of blocked DNA ends by at least two distinct mechanisms. First, DNA damage and cell cycle-dependent phosphorylation leads to Sae2 tetramerization. Second, and independently, phosphorylation of the conserved C-terminal domain of Sae2 is a prerequisite for its physical interaction with Rad50, which is also crucial to promote the MRX endonuclease. The lack of this interaction explains the phenotype of rad50S mutants defective in the processing of Spo11-bound DNA ends during meiotic recombination. Our results define how phosphorylation controls the initiation of DNA end resection and therefore the choice between the key DNA double-strand break repair mechanisms.
Highlights
DNA end resection plays a critical function in DNA double-strand break repair pathway choice
We show that phosphorylation at multiple sites promotes the formation of active Sae[2] tetramers and regulates a physical interaction with the Rad[50] subunit of the MRX complex
We show that pSae[2] that had been dephosphorylated by λ phosphatase can be subsequently partially activated upon phosphorylation by human recombinant CDK1/ Cyclin B in vitro (Fig. 3f, g)
Summary
DNA end resection plays a critical function in DNA double-strand break repair pathway choice. Independently, phosphorylation of the conserved C-terminal domain of Sae[2] is a prerequisite for its physical interaction with Rad[50], which is crucial to promote the MRX endonuclease The lack of this interaction explains the phenotype of rad50S mutants defective in the processing of Spo11-bound DNA ends during meiotic recombination. Our results define how phosphorylation controls the initiation of DNA end resection and the choice between the key DNA double-strand break repair mechanisms. The resection and resulting recombinational repair of meiotic DSBs absolutely require Sae[2] and MRX and their orthologs, because Spo[11] remains covalently bound to the break ends and needs to be removed by MRX and Sae[214–17] This stands in contrast to resection in yeast vegetative cells, which can be partially MRX-Sae[2] independent[18,19]. Our results provide a mechanistic basis for the regulatory control of DSB repair pathway choice by Sae[2] phosphorylation
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