Alternative Mechanisms for Coordinating Polymerase α and MCM Helicase

Abstract

Functional coordination between DNA replication helicases and DNA polymerases at replication forks, achieved through physical linkages, has been demonstrated in prokaryotes but not in eukaryotes. In Saccharomyces cerevisiae, we showed that mutations that compromise the activity of the MCM helicase enhance the physical stability of DNA polymerase α in the absence of their presumed linker, Mcm10. Mcm10 is an essential DNA replication protein implicated in the stable assembly of the replisome by virtue of its interaction with the MCM2-7 helicase and Polα. Dominant mcm2 suppressors of mcm10 mutants restore viability by restoring the stability of Polα without restoring the stability of Mcm10, in a Mec1-dependent manner. In this process, the single-stranded DNA accumulation observed in the mcm10 mutant is suppressed. The activities of key checkpoint regulators known to be important for replication fork stabilization contribute to the efficiency of suppression. These results suggest that Mcm10 plays two important roles as a linker of the MCM helicase and Polα at the elongating replication fork—first, to coordinate the activities of these two molecular motors, and second, to ensure their physical stability and the integrity of the replication fork.

We thank a very talented group of undergraduate and rotation students, Alice Leung, Mike Singer, Jany Chan, and Stephanie Yazinski, for identifying many of the suppressor alleles. We thank Nozomi Sakakibara for her help in the construction of the Mth mutants. We also thank Marcus Smolka and Francisco M. Bastos de Oliviera for their intellectual and technical input, especially with regard to checkpoint effects. Lastly, we thank the Huberman lab for their patience and time in teaching C.L. the 2D gel technique.

This work was supported by NSF (MBG-0453773) and NIH GM072557 awarded to B.K.T. and NSF (MCB-0815646) awarded to Z.K.