Physical Interactions between Mcm10, DNA, and DNA Polymerase α

Abstract

Mcm10 is an essential eukaryotic protein required for the initiation and elongation phases of chromosomal replication. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase ␣ (pol ␣), with chromatin. We showed previously that the internal (ID) and C-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss) DNA and the catalytic p180 subunit of pol ␣. However, the mechanism by which Mcm10 interacts with pol ␣ on and off DNA is unclear. As a first step toward understanding the structural details for these critical intermolecular interactions, x-ray crystallography and NMR spectroscopy were used to map the binary interfaces between Mcm10-ID, ssDNA, and p180. The crystal structure of an Mcm10-ID⅐ssDNA complex confirmed and extended our previous evidence that ssDNA binds within the oligonucleotide/oligosaccharide binding-fold cleft of Mcm10-ID. We show using NMR chemical shift perturbation and fluorescence spectroscopy that p180 also binds to the OB-fold and that ssDNA and p180 compete for binding to this motif. In addition, we map a minimal Mcm10 binding site on p180 to a small region within the p180 N-terminal domain (residues 286 -310). These findings, together with data for DNA and p180 binding to an Mcm10 construct that contains both the ID and CTD, provide the first mechanistic insight into how Mcm10 might use a handoff mechanism to load and stabilize pol ␣ within the replication fork.

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

“…21,22,[24][25][26][27][28][29][30][31] Therefore, our data are consistent with the notion that inefficient chromatin assembly of pol-α/primase complexes in both pol1-1 and mcm10-1 mutants may be responsible for the impairment of DNA synthesis, which triggers DRIM. However, we cannot exclude that Mcm10 depletion interferes with DNA synthesis in a manner unrelated to priming.…”

“…[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.…”

Multiple Functions for Drosophila Mcm10 Suggested Through Analysis of Two Mcm10 Mutant Alleles

“…The highly conserved core with its single atypical zinc-finger domain is present in all eukaryotes examined to date and likely represents the most ancient function of this protein. This core has been shown to interact with single-stranded DNA, DNA polymerase a, and PCNA Chattopadhyay and Bielinsky 2007;Robertson et al 2008;Warren et al 2008Warren et al , 2009). Genetic studies also support the assertion that this central core supports the essential function(s) of Mcm10 as mutations in this region of Mcm10 affect viability of cells and affect DNA replication (Merchant et al 1997;Homesley et al 2000;Ricke and Bielinsky 2006).…”

“…As well as the core, higher eukaryotes also have another conserved domain at the C terminus of the protein that contains two additional zinc-finger motifs (Robertson et al 2008;Warren et al 2008Warren et al , 2009). The observation that this additional domain is present only in higher eukaryotes suggests that Mcm10 has taken on additional roles that are mediated through this Cterminal domain.…”

“…Alignments from multiple species reveal that the Mcm10 protein contains several conserved regions ( Figure 1A). The central highly conserved region of Mcm10 is present in all Mcm10 proteins and possesses an essential zinc-finger domain that has been implicated in protein-protein interactions (Robertson et al 2008;Warren et al 2008Warren et al , 2009). In addition to this conserved core, higher eukaryotic Mcm10 proteins have expanded in length and contain a highly conserved C-terminal domain with two zinc-finger motifs (Robertson et al 2008;Warren et al 2008Warren et al , 2009.…”