| Autor: |
Robertson PD; Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232., Warren EM; Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232., Zhang H; Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232., Friedman DB; Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232., Lary JW; National Analytical Ultracentrifugation Facility, University of Connecticut, Storrs, Connecticut 06269., Cole JL; National Analytical Ultracentrifugation Facility, University of Connecticut, Storrs, Connecticut 06269., Tutter AV; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115., Walter JC; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115., Fanning E; Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232., Eichman BF; Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232; Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232. Electronic address: brandt.eichman@vanderbilt.edu. |
| Abstrakt: |
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 alpha-primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase alpha-primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome. |
