Effects of CDC45 mutations on DNA replication and genome stability

Abstract

Cdc45 is a non-catalytic subunit of the CMG helicase complex that is recruited to the autonomously replicating sequence at the onset of DNA replication. The Cdc45 protein is required for the initiation of DNA replication as well as for nascent DNA strand synthesis. It interacts with Mcm2 and Psf1 elements of CMG helicase, as well as with Sld3, an initiation factor, and Pol2, the catalytic subunit of DNA polymerase epsilon (Pol ε). In this study, we analyzed the effects of amino acid substitutions in the Cdc45 region involved in the interaction of this protein with Mcm2–7 (Cdc45-1), Psf1 (Cdc45-26), and Sld3 (Cdc45-25, Cdc45-35). We found that mutations in CDC45 resulted in defective DNA replication. Under permissive conditions, delayed DNA synthesis was observed. At restrictive temperatures, the mutant cells were unable to efficiently replicate DNA. However, after the initiation of DNA replication under permissive conditions, the four analyzed CDC45 mutants exhibited DNA synthesis under the restrictive conditions. Moreover, we observed increased mutation rates, mainly dependent on DNA polymerase zeta (Pol ζ), as well as increased incidence of replication errors. These findings confirm the essential function of Cdc45 in DNA replication initiation and demonstrate that impaired Cdc45 subunit has an impact on the fidelity of the nascent DNA strand synthesis. The changes in cell function observed in this study, related to defects in Cdc45 function, may help understand some diseases associated with CDC45.