<em>Saccharomyces cerevisiae</em> Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks
Supplementary Material
Abstract
Single-stranded DNA constitutes an important early intermediate for homologous recombination and damage-induced cell cycle checkpoint activation. In Saccharomyces cerevisiae, efficient double-strand break (DSB) end resection requires several enzymes; Mre11/Rad50/Xrs2 (MRX) and Sae2 are implicated in the onset of 5′-strand resection, whereas Sgs1/Top3/Rmi1 with Dna2 and Exo1 are involved in extensive resection. However, the molecular events leading to a switch from the MRX/Sae2-dependent initiation to the Exo1- and Dna2-dependent resection remain unclear. Here, we show that MRX recruits Dna2 nuclease to DSB ends. MRX also stimulates recruitment of Exo1 and antagonizes excess binding of the Ku complex to DSB ends. Using resection assay with purified enzymes in vitro, we found that Ku and MRX regulate the nuclease activity of Exo1 in an opposite way. Efficient loading of Dna2 and Exo1 requires neither Sae2 nor Mre11 nuclease activities. However, Mre11 nuclease activity is essential for resection in the absence of extensive resection enzymes. The results provide new insights into how MRX catalyses end resection and recombination initiation.
Acknowledgments
We thank S Brill, J Haber, J Campbell and A Tomkinson for gifts of strains, plasmids, antibodies and purified proteins, and D Villarreal for an editorial help. We also thank the members of the SEL, TTP and GI laboratory for helpful discussion. This work is funded by grants from NIH to SEL (GM071011, 3R01 GM071011), TTP (CA094008) and GI (GM080600 and 3R01GM080600). SEL is a scholar of the Leukemia and Lymphoma Society. Studies performed in the Paull laboratory were also supported by the Howard Hughes Medical Institute.
Footnotes
The authors declare that they have no conflict of interest.