The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum ΔH contains DNA helicase activity
- Program of Molecular Biology, Memorial Sloan–Kettering Cancer Center, 1275 York Avenue, Box 97, New York, NY 10021
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Contributed by Jerard Hurwitz
Abstract
Previous studies have identified an ATP-dependent DNA helicase activity intrinsic to the human minichromosome maintenance (MCM) complex, composed of MCM subunits 4, 6, and 7 [Ishimi, Y. (1997) J. Biol. Chem. 272, 24508–24513]. In contrast to the presence of multiple MCM genes (at least six) in eukaryotes, the archaeon Methanobacterium thermoautotrophicum ΔH (mth) genome contains a single open reading frame coding for an MCM protein. In this study we report the isolation of the mthMCM protein overexpressed in Escherichia coli. The purified recombinant protein was found to exist in both multimeric (≈103 kDa) and monomeric (76 kDa) forms. Both forms of the protein bind to single-stranded DNA, hydrolyze ATP in the presence of DNA, and possess 3′-to-5′ ATP-dependent DNA helicase activities. Thus, a single mthMCM protein contains biochemical properties identical to those associated with the eukaryotic MCM4, -6, and -7 complex. These results suggest that the characterization of the mthMCM protein and its multiple forms may contribute to our understanding of the role of MCM helicase activity in eukaryotic chromosomal DNA replication.
Footnotes
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↵ * Z.K. and J.-K.L. contributed equally to this work.
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↵ † To whom reprint requests should be addressed. E-mail: j-hurwitz{at}ski.mskcc.org.
- Abbreviations:
- ORC,
- origin recognition complex;
- sp-,
- Schizosaccharomyces pombe;
- h-,
- human;
- MCM,
- minichromosome maintenance;
- mth-,
- Methanobacterium thermoautotrophicum ΔH;
- ss,
- single-stranded;
- ds,
- double-stranded;
- T Ag,
- simian virus 40 large tumor antigen;
- SSB,
- ssDNA-binding protein;
- RPA,
- replication protein A;
- ATP-γ-S,
- γ-thio-ATP
- Copyright © 1999, The National Academy of Sciences
