Domain Architecture and Biochemical Characterization of Vertebrate Mcm10

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 biochemica...

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Bibliographic Details
Published in:The Journal of biological chemistry 2008-02, Vol.283 (6), p.3338-3348
Main Authors: Robertson, Patrick D., Warren, Eric M., Zhang, Haijiang, Friedman, David B., Lary, Jeffrey W., Cole, James L., Tutter, Antonin V., Walter, Johannes C., Fanning, Ellen, Eichman, Brandt F.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Anisotropy
Cell Cycle Proteins - chemistry
Cell Cycle Proteins - physiology
DNA Replication
DNA-Binding Proteins - chemistry
DNA-Binding Proteins - physiology
DNA-Directed DNA Polymerase - chemistry
Edetic Acid - pharmacology
Humans
Minichromosome Maintenance Proteins
Molecular Sequence Data
Protein Binding
Protein Conformation
Protein Structure, Tertiary
Spectrometry, Fluorescence - methods
Xenopus laevis
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Summary: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 α-primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase α-primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M706267200