Studies of the cell cycle regulation of checkpoint activation and DNA double -strand break repair in Saccharomyces cerevisiae.

摘要

Efficient recognition and repair of DNA lesions governs genome integrity and cell survival. Different DNA lesions are repaired via distinct pathways and the manner in which lesions are recognized and processed for repair is dictated by the cell cycle. DNA double-strand breaks (DSBs) are potentially lethal lesions that can be repaired using either non-homologous end joining (NHEJ) or homologous recombination (HR). In the budding yeast Saccharomyces cerevisiae, these lesions are recognized by the DNA checkpoint machinery through the association of the Mre11-Rad50-Xrs2 (MRX) complex with the exposed DNA ends as well as by the binding of single stranded DNA (ssDNA) tails by replication protein A (RPA). MRX binds and recognizes DNA ends at every stage of the cell cycle, promoting NHEJ at DNA ends also bound by the Yku70/Yku80 heterodimer in G1 cells. DNA ends not suitable for NHEJ are processed by MRX-Sae2 and other nucleases to form ssDNA tails bound by RPA in both G1 and S/G2 cells. The central homologous recombination protein Rad52 can only be recruited to the RPA-coated ssDNA once the cell has entered S phase and the B-type cyclins are present. During DNA replication, Mec1 inhibits Rad52 focus formation at the sites of stalled DNA replication forks and at DSBs when the intra-S phase checkpoint has been activated. Thus, three major steps in DSB repair are cell cycle regulated: DNA end processing, the creation of ssDNA and the recruitment of the homologous recombination machinery.;The DNA checkpoint kinases Mec1 and Tel1 are also cell cycle regulated. Tel1 is activated in response to DNA damage that is recognized by the NHEJ protein Yku70 at both DNA DSBs and at telomeres while the Mec1/Ddc2 kinase is activated by the DNA damage clamp 9-1-1. The 9-1-1 complex is also required to localize Mec1/Ddc2 to the sites of DNA damage in G1 cells while RPA independently recruits Mec1/Ddc2 in response to DNA damage in S/G2 cells. Thus, the DNA checkpoint machinery can be activated through different pathways depending on the stage of the cell cycle. Together, these studies demonstrate the importance of the B-type cyclins and entry into S phase for both full checkpoint activation and the recruitment of the homologous recombination machinery in response to DNA damage.