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.

In yeast, Rif1 is part of the telosome, where it inhibits telomerase and checkpoint signaling at chromosome ends. In mammalian cells, Rif1 is not telomeric, but it suppresses DNA end resection at chromosomal breaks, promoting repair by nonhomologous end joining (NHEJ). Here, we describe crystal structures for the uncharacterized and conserved ∼125-kDa N-terminal domain of Rif1 from Saccharomyces cerevisiae (Rif1-NTD), revealing an α-helical fold shaped like a shepherd's crook. We identify a high-affinity DNA-binding site in the Rif1-NTD that fully encases DNA as a head-to-tail dimer. Engagement of the Rif1-NTD with telomeres proved essential for checkpoint control and telomere length regulation. Unexpectedly, Rif1-NTD also promoted NHEJ at DNA breaks in yeast, revealing a conserved role of Rif1 in DNA repair. We propose that tight associations between the Rif1-NTD and DNA gate access of processing factors to DNA ends, enabling Rif1 to mediate diverse telomere maintenance and DNA repair functions.

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