Helicase CMG and Mph1 involved in two distinct steps of DNA replication

Abstract

Mcm2-7, ring-structured complex composed of six different subunits, was postulated as a replicative helicase in eukaryotes. Although it doesn’t have helicase activity in vitro, many data indirectly support that it would unwind duplex DNA ahead of replication fork in DNA replication. Recently, the complex containing Cdc45, Mcm2-7 and GINS that has helicase activity was isolated from Drosophila. Based on that report, for the purpose of reconstitution of eukaryotic replication system in vitro, human CMG (Cdc45/Mcm2-7/GINS) complex was isolated in this study. It showed 3’ to 5’ helicase and the ATPase activities. In addition, this helicase activity was stimulated by fork-structured substrate. Mutated versions of CMG complex in putative ATP motif of Mcm subunits showed reduced helicase activity. All these data suggest that CMG complex is universal replicative helicase in eukaryotes. In the course of lagging strand synthesis, the 5’ end of downstream Okazaki fragment is displaced, referred as flap-structure, and cleaved by two structure-specific endonucleases Dna2 and Fen1 to make ligatable nicks. In this study, to find novel factors participate in Okazaki fragment processing, multicopy suppressor screens were performed with Saccharomyces cerevisiae dna2K1080E helicase-negative lethal mutant and it resulted in the isolation of MPH1 gene. Purified Mph1 stimulated the endonuclease activities of both Fen1 and Dna2. This stimulation required neither ATP hydrolysis nor the helicase activity of Mph1. The stimulation of Fen1 by Mph1 was limited to flap-structured substrates through its structure-specific binding activity. Our biochemical and genetic data indicate that Mph1 plays an important, although not essential, role in processing of Okazaki fragments by facilitating formation of ligatable nicks through the stimulation of flap-cleaving enzymes. The Mph1 is basically 3’ to 5’ helicase, however it showed unwinding activities on a variety of substrate with highly sequ...