Journal of Biological Chemistry
Volume 284, Issue 36, 4 September 2009, Pages 24662-24672
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DNA: Replication, Repair, Recombination, and Chromosome Dynamics
Physical Interactions between Mcm10, DNA, and DNA Polymerase α*

https://doi.org/10.1074/jbc.M109.020438Get rights and content
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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 α (pol α), 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 α. However, the mechanism by which Mcm10 interacts with pol α 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-ID·ssDNA 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 α within the replication fork.

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The atomic coordinates and structure factors (code 3H15) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

*

This work was supported, in whole or in part, by National Institutes of Health Grants R01 GM080570 (to B. F. E.), R01 GM65484 (to W. J. C.), and R01 GM52948 (to E. F.). Additional support for crystallography and mass spectrometry facilities was provided by the Vanderbilt Center in Molecular Toxicology (National Institutes of Health Grant P30 ES000267) and the Vanderbilt-Ingram Cancer Center (National Institutes of Health Grant P30 CA068485).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S9.

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Supported in part by Molecular Biophysics Training Grant T32 GM08320.