Domain architecture and biochemical characterization of vertebrate Mcm10

J Biol Chem. 2008 Feb 8;283(6):3338-3348. doi: 10.1074/jbc.M706267200. Epub 2007 Dec 6.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Mcm10 protein, Xenopus
  • Edetic Acid
  • DNA-Directed DNA Polymerase
  • Minichromosome Maintenance Proteins