Mcm10 Self-Association Is Mediated by an N-Terminal Coiled-Coil Domain

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.

MCM10: One tool for all—Integrity, maintenance and damage control

“…Semi-quantitative chromatin binding studies are consistent with recent findings that revealed that the conserved CC domain in the NTD enables XMcm10 to dimer- and trimerize in a highly dynamic fashion [5, 8, 15]. Removal of the corresponding motif in ScMcm10 leads to severe hydroxyurea (HU) sensitivity when checkpoint function is compromised [8](Alver and Bielinsky, unpublished). The NTD has also been implicated in oligomerization of human (Hs) Mcm10 [16].…”

“…Several reports suggest that Mcm10 has a strong preference for ss over ds DNA [10, 12, 13], providing a rationale of how Mcm10 might contribute to the activation of the Cdc45:Mcm2-7:GINS (CMG) helicase [14]. Semi-quantitative chromatin binding studies are consistent with recent findings that revealed that the conserved CC domain in the NTD enables XMcm10 to dimer- and trimerize in a highly dynamic fashion [5, 8, 15]. Removal of the corresponding motif in ScMcm10 leads to severe hydroxyurea (HU) sensitivity when checkpoint function is compromised [8](Alver and Bielinsky, unpublished).…”

“…The ID is flanked by an N-terminal domain (NTD), which displays a coiled coil (CC) motif of considerable sequence similarity among species [7, 8]. In contrast, the C-terminal domain (CTD) varies highly from uni- to multicellular organisms and is distinguished by a large metazoan-specific extension [9].…”

“…Independent studies of Schizosaccharomyces pombe (Sp) and Xenopus leavis (X) Mcm10 implicated the NTD in self-interaction [8, 10, 11]. Further support for oligomerization came from in vitro studies that characterized the binding of ScMcm10 to single-stranded (ss) DNA, which estimated that a Mcm10-trimer occupied up to 50 nucleotides [12].…”

The N-terminus of Mcm10 is important for interaction with the 9-1-1 clamp and in resistance to DNA damage

“…Toward this end, we constructed N-terminal truncation mutants of Mcm10 that left the PIP box intact, but deleted the first 50, 100 or 150 amino acids (Figure 2A). This region of Mcm10 lies outside the conserved OB-fold/Zn-finger domain and is not predicted to have any significant secondary structure (17), except for a coiled-coil region at the very N-terminus (21). All fusion proteins were expressed (Figure 2B and Supplementary Figure S1) and we then assessed binding to Mec3 via two-hybrid analyses (Figure 2C).…”