Isolation and Characterization of Various Complexes of the Minichromosome Maintenance Proteins of Schizosaccharomyces pombe*
- From the Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Abstract
Minichromosome maintenance (Mcm) proteins 2–7 are highly conserved in eukaryotes and play an essential role in DNA replication. Here, we describe the reconstitution of the various complexes of the Mcm proteins of Schizosaccharomyces pombeusing the baculovirus expression system. The simultaneous expression of all six of the Mcm proteins, as well as different combinations of these proteins, yielded several stable complexes that included the heterohexamer of Mcm2/3/4/5/6/7, the Mcm2/4/6/7 heterotetramer, the dimer of the Mcm4/6/7 heterotrimer, and the Mcm3/5 heterodimer. The purification and characterization of the biochemical properties of these complexes showed that only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3′ to 5′ DNA helicase activities. Consistent with these results, the interaction of either Mcm2 or Mcm3/5 with the Mcm4/6/7 complex resulted in the disassembly of the dimeric complex of Mcm4/6/7 and the loss of DNA helicase activity. These results suggest that the Mcm4/6/7 complex is a catalytic core of the Mcm complex and that Mcm2 and Mcm3/5 may be involved in the regulation of the activity of this complex.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant GM 58559.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed. Fax: 212-717-3627; E-mail: j-hurwitz@ski.mskcc.org.
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Published, JBC Papers in Press, April 18, 2000, DOI 10.1074/jbc.M001118200
- Abbreviations:
- MCM
-
minichromosome maintenance
- ss
-
single stranded
- ORC
-
origin recognition complex
- mth
-
Methanobacterium thermoautotrophicum
- T Ag
-
simian virus 40 large tumor antigen
- PCR
-
polymerase chain reaction
- BSA
-
bovine serum albumin
- sp
-
S. pombe
- SSB
-
single stranded-binding protein
- Ab
-
antibody
- PAGE
-
polyacrylamide gel electrophoresis
- DTT
-
dithiothreitol
- ATPγS
-
adenosine 5′-O-(thiotriphosphate)
-
- Received February 9, 2000.
- Revision received March 30, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
