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J. Biol. Chem., Vol. 283, Issue 6, 3338-3348, February 8, 2008

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Domain Architecture and Biochemical Characterization of Vertebrate Mcm10^*

Patrick D. Robertson¹, Eric M. Warren¹², Haijiang Zhang¹, David B. Friedman, Jeffrey W. Lary^¶, James L. Cole^¶, Antonin V. Tutter^||, Johannes C. Walter^||, Ellen Fanning, and Brandt F. Eichman^**3

From the Department of Biological Sciences, Department of Biochemistry, and ^**Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232, ^¶National Analytical Ultracentrifugation Facility, University of Connecticut, Storrs, Connecticut 06269, and ^||Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Mcm10 plays a key role in initiation and elongation of eukaryotic chromosomal DNA replication. As a first step to better understand the structure and function of vertebrate Mcm10, we have determined the structural architecture of Xenopus laevis Mcm10 (xMcm10) and characterized each domain biochemically. Limited proteolytic digestion of the full-length protein revealed N-terminal-, internal (ID)-, and C-terminal (CTD)-structured domains. Analytical ultracentrifugation revealed that xMcm10 self-associates and that the N-terminal domain forms homodimeric assemblies. DNA binding activity of xMcm10 was mapped to the ID and CTD, each of which binds to single- and double-stranded DNA with low micromolar affinity. The structural integrity of xMcm10-ID and CTD is dependent on the presence of bound zinc, which was experimentally verified by atomic absorption spectroscopy and proteolysis protection assays. The ID and CTD also bind independently to the N-terminal 323 residues of the p180 subunit of DNA polymerase -primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase -primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.

Received for publication, July 30, 2007 , and in revised form, December 4, 2007.

^* This work was funded by National Institutes of Health Grants GM080570 (to B. F. E.) and GM52948 (to E. F.) and by the Vanderbilt Discovery Grant Program. Additional support for facilities came from the Vanderbilt Center in Molecular Toxicology Grant P30 ES000267. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5.

¹ These authors contributed equally to this work.

² Supported from the Molecular Biophysics Training Grant T32 GM08320.

³ To whom correspondence should be addressed: VU Station B, Box 35-1634, Nashville, TN 37235-1634. Fax: 615-343-6707; E-mail: brandt.eichman{at}vanderbilt.edu.

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