Alternative Mechanisms for Coordinating Polymerase and MCM Helicase

Department of Molecular Biology & Genetics, Cornell University, Ithaca, New York 14853, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 11/2009; 30(2):423-35. DOI: 10.1128/MCB.01240-09
Source: PubMed


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.

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    • "Mcm10 FL and Mcm2 FL Two hybrid (Lee et al. 2010) Mcm10 FL and Mcm3 FL Two hybrid (Liachko and Tye 2009) Mcm10 FL and Mcm6 FL Two hybrid (Merchant et al. 1997) Mcm10 FL and Mcm6 FL Two hybrid (van Deursen et al. 2012) Chromosoma seems to activate the complex, since the CMG cannot unwind DNA in the absence of Mcm10 and stays associated with the origin (Kanke et al. 2012; van Deursen et al. 2012; Watase et al. 2012). Finally, with the help of Ctf4, DNA polymerase α (Polα) is recruited (Gambus et al. 2009; Kang et al. 2013; Simon et al. 2014). "
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    ABSTRACT: A crucial step during eukaryotic initiation of DNA replication is the correct loading and activation of the replicative DNA helicase, which ensures that each replication origin fires only once. Unregulated DNA helicase loading and activation, as it occurs in cancer, can cause severe DNA damage and genomic instability. The essential mini-chromosome maintenance proteins 2-7 (MCM2-7) represent the core of the eukaryotic replicative helicase that is loaded at DNA replication origins during G1-phase of the cell cycle. The MCM2-7 helicase activity, however, is only triggered during S-phase once the holo-helicase Cdc45-MCM2-7-GINS (CMG) has been formed. A large number of factors and several kinases interact and contribute to CMG formation and helicase activation, though the exact mechanisms remain unclear. Crucially, upon DNA damage, this reaction is temporarily halted to ensure genome integrity. Here, we review the current understanding of helicase activation; we focus on protein interactions during CMG formation, discuss structural changes during helicase activation, and outline similarities and differences of the prokaryotic and eukaryotic helicase activation process.
    Chromosoma 10/2014; 124(1). DOI:10.1007/s00412-014-0489-2 · 4.60 Impact Factor
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    • "It accomplishes this through its interactions with several components of the core replication machinery, including the Mcm2-7 helicase (11), polymerase-α (Polα)/primase (3,12–14) and the replication clamp, proliferating cell nuclear antigen (PCNA) (15). Loss of Mcm10 protein expression results in induction of DNA breaks as well as checkpoint activation (3,16). "
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    ABSTRACT: Accurate replication of the genome requires the evolutionarily conserved minichromosome maintenance protein, Mcm10. Although the details of the precise role of Mcm10 in DNA replication are still debated, it interacts with the Mcm2-7 core helicase, the lagging strand polymerase, DNA polymerase-α and the replication clamp, proliferating cell nuclear antigen. Loss of these interactions caused by the depletion of Mcm10 leads to chromosome breakage and cell cycle checkpoint activation. However, whether Mcm10 has an active role in DNA damage prevention is unknown. Here, we present data that establish a novel role of the N-terminus of Mcm10 in resisting DNA damage. We show that Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress evoked by UV irradiation or nucleotide shortage. We map the interaction domain with Mec3 within the N-terminal region of Mcm10 and demonstrate that its truncation causes UV light sensitivity. This sensitivity is not further enhanced by a deletion of MEC3, arguing that MCM10 and MEC3 operate in the same pathway. Since Rad53 phosphorylation in response to UV light appears to be normal in N-terminally truncated mcm10 mutants, we propose that Mcm10 may have a role in replication fork restart or DNA repair.
    Nucleic Acids Research 06/2014; 42(13). DOI:10.1093/nar/gku479 · 9.11 Impact Factor
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    • "Mcm10 interacts with single- (ss) and double-stranded (ds) DNA [19], [20], [21], is loaded onto chromatin in early S-phase, and is essential for helicase activation [22], [23], [24] and the subsequent recruitment of replisome proteins, including RPA and pol α [4], [16], [25]. Saccharomyces cerevisiae Mcm10 (scMcm10) is required to maintain pol α on chromatin independently of Cdc45 [16], and both Mcm10 and the sister chromatid cohesion protein And-1/Ctf4 have been implicated in loading pol α onto chromatin and physically coupling pol α and Mcm2–7 [15], [16], [18], [26], [27]. Mcm10 from various organisms has been shown to interact physically with key proteins involved in initiation and elongation, including ORC [28], [29], Mcm2–7 [28], [30], [31], [32], pol α [16], [17], [20], [33], [34], proliferating cell nuclear antigen (PCNA) [35], And-1 [18] and the RecQ-like helicase RecQ4 [18], [36]. "
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    ABSTRACT: Minichromosome maintenance protein 10 (Mcm10) is an essential eukaryotic DNA-binding replication factor thought to serve as a scaffold to coordinate enzymatic activities within the replisome. Mcm10 appears to function as an oligomer rather than in its monomeric form (or rather than as a monomer). However, various orthologs have been found to contain 1, 2, 3, 4, or 6 subunits and thus, this issue has remained controversial. Here, we show that self-association of Xenopus laevis Mcm10 is mediated by a conserved coiled-coil (CC) motif within the N-terminal domain (NTD). Crystallographic analysis of the CC at 2.4 Å resolution revealed a three-helix bundle, consistent with the formation of both dimeric and trimeric Mcm10 CCs in solution. Mutation of the side chains at the subunit interface disrupted in vitro dimerization of both the CC and the NTD as monitored by analytical ultracentrifugation. In addition, the same mutations also impeded self-interaction of the full-length protein in vivo, as measured by yeast-two hybrid assays. We conclude that Mcm10 likely forms dimers or trimers to promote its diverse functions during DNA replication.
    PLoS ONE 07/2013; 8(7):e70518. DOI:10.1371/journal.pone.0070518 · 3.23 Impact Factor
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