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 ␣ 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 Pol␣. Dominant mcm2 suppressors of mcm10 mutants restore viability by restoring the stability of Pol␣ 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 Pol␣ 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.The key players of the replication machinery are the DNA polymerases that synthesize the leading and lagging daughter strands and the replicative helicase that unwinds the parental strands ahead of the polymerases. Coordination between the helicase and the polymerases is critical during replication. Uncoupling of these two molecular machines, especially during lagging strand synthesis, may result in an unrestrained helicase and the exposure of extensive single-stranded DNA (ssDNA), as observed in checkpoint mutants treated with hydroxyurea (HU) (37). Although there is no direct evidence, the implication is that the replicative helicase would be moving at a faster pace than would the DNA polymerase if synchrony were destroyed. In Escherichia coli, the replicative helicase (DnaB) and the primase (DnaG) are coupled by direct contact to form a tight complex (3). In T7, processivity of the gp5 polymerase in lagging strand synthesis requires coupling to the gp4 helicase (16). Recent studies of the budding yeast Saccharomyces cerevisiae suggest that Mrc1 may couple DNA polymerase ε and the MCM helicase on the leading strand as well as activate the checkpoint response under replication stress (1,22,28). A candidate for coupling DNA polymerase ␣ primase and the MCM helicase on the lagging strand is Mcm10, because Mcm10 interacts with subunits of the Mcm2-7 helicase (26, 29) as well as Pol␣ (14, 33) and the stability of Pol␣ requires Mcm10 in both budding yeast and human cells (8,33). Mcm10 is an essential protein known to be involved in various aspects of the replication process. It is required during both initiation and elongation steps of DNA replication and interacts with a wide range of replication factors, such as ORC (17,23,29), MCM helicase, DNA polymerases ε and ␦ (23), Cdc45 (...

Mcm10 deficiency causes defective-replisome-induced mutagenesis and a dependency on error-free postreplicative repair

“…21,23,25,28 Importantly, we also demonstrate that disruption of Mcm10 function leads to pol-ζ-dependent DRIM. This is particularly relevant to cancer biology, because dysregulation of translesion polymerases is strongly associated with tumor formation [68][69][70][71][72][73] and negative clinical outcomes.…”

“…[21][22][23][24][25][26][27][28][29][30] Moreover, Mcm10 is post-translationally modified during G 1 and S phase, resulting in non-proteolytic ubiquitination at 2 distinct lysines. This modification is a prerequisite for Mcm10's interaction with PCNA, which is essential for cell proliferation.…”

From structure to mechanism—understanding initiation of DNA replication

“…Therefore, the two proteins might affect Mcm2 in alternative ways, with Cdc45 closing the gate to support helicase activity, and Cdt1 allowing opening and closing of the gate during MCM2-7 loading. Moreover, Mcm10 might affect these surfaces as well, as it contacts the Mcm2 NTD (Apger et al 2010;Lee et al 2010;Looke et al 2017) in order to stabilize the CMG complex (Looke et al 2017). At an in vitro assembled replication fork, ssDNA appears from the N-terminal MCM2-7 face during DNA unwinding (Georgescu et al 2017).…”

Cell Cycle Control of DNA Replication by Phosphorylation and Dephosphorylation of Replication-Initiation Proteins in Budding Yeast

“…Budding yeast Mcm10p is a recruitment factor that targets polymerase α to the licensed origins in G1 phase and the replication forks during S-phase (Ricke & Bielinsky, 2004;Lee et al, 2010). It binds to origins in G1 cells in an MCM-dependent manner and co-migrates with the fork as part of the replisome progression complex (RPC) (Gambus et al, 2006).…”