Cdc7-dependent interactions of Mcm2-7 with G1-loaded Rad51 and Rad52 regulate the recombinational response to replicative damage

Abstract

The response to DNA lesions that impair the advance of replication forks relies on DNA damage tolerance (DDT) mechanisms, which facilitate fork bypass across the lesions and repair of the ssDNA fragments generated during this process. One of these mechanisms is Homologous Recombination (HR), which uses the information of an intact template to repair the ssDNA fragments. An important mechanistic difference with the replication-independent process of double-strand break (DSB) repair by HR is that Rad52 and Rad51 travel with the forks and their loading into the ssDNA lesions is coupled to DNA replication. We show in Saccharomyces cerevisiae that Rad51 interacts physically with the replicative helicase MCM2-7 regardless of the presence of DNA damage, and that this interaction requires the continuous activity during S phase of the cell-cycle master kinase Cdc7. Remarkably, even though Rad51 and MCM2-7 are recruited independently to chromatin, they display similar kinetics of DNA binding during the cell cycle. In the absence of DNA damage they accumulate in G1 and are released as replication is completed, whereas in the presence of the alkylating agent methyl-methane sulfonate (MMS) they remain bound to chromatin, suggesting that the helicase MCM2-7 plays a role in DDT. The loading/stabilization of Rad51 in response to MMS requires the kinase activity of Cdc7. Likewise, the binding of Rad52 to ssDNA lesions generated upon treatment with MMS is impaired in a thermosensitive cdc7-4 allele at semi-permissive temperature, being this effect independent of the roles of Cdc7 on replication initiation, checkpoint activation or translesion synthesis. Instead, it is partially mediated by phosphorylation of Mcm2 at serines 164 and 170. However, neither cdc7-4 nor a phosphomimetic mcm2AA affect the association between Rad51 and MCM2-7, suggesting that Cdc7 maintains Rad52/Rad51/MCM2-7 at chromatin in response to DNA damage through a different mechanism. Consistent with these results a reduction in the kinase activity of Cdc7 affects the kinetics of sister-chromatid junctions and Rad52 repair in response to MMS. Overall, these results uncover a novel role for Cdc7 in the recombinational branch of DDT by regulating the binding of Rad51 to chromatin in coordination with the helicase MCM2-7.