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J Biol Chem, Vol. 275, Issue 10, 7327-7336, March 10, 2000

A Unique Organization of the Protein Subunits of the DNA Polymerase Clamp Loader in the Archaeon Methanobacterium thermoautotrophicum Delta H*

Zvi KelmanDagger and Jerard Hurwitz§

From the Department of Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Replication factor C (RFC, also called activator 1), in conjunction with proliferating cell nuclear antigen (PCNA), is responsible for processive DNA synthesis catalyzed by the eukaryotic replicative DNA polymerases delta  and epsilon . Here we report the isolation and characterization of homologues of RFC and PCNA from the archaeon, Methanobacterium thermoautotrophicum Delta H. In contrast to the five subunit RFC complex isolated from eukaryotic cells, the mthRFC contains only two subunits. The two genes encoding the RFC subunits called, mthRFC1 and mthRFC3, were cloned, and the proteins (54.4 and 36.8 kDa, respectively) were overexpressed in Escherichia coli and purified individually and as a complex. The gene encoding PCNA was also cloned, and the protein was purified after overexpression in E. coli. Based on sizing column elution and subunit composition, the mthRFC complex appears to be a hexamer consisting of two mthRFC1 protomers and four mthRFC3 protomers. Although mthRFC differs in organization from its eukaryotic counterpart, it was shown to be functionally similar to eukaryotic RFC in: (i) catalyzing DNA-dependent ATP hydrolysis; (ii) binding preferentially to DNA primer ends; (iii) loading mthPCNA onto singly nicked circular DNA; and (iv) supporting mthPolB-catalyzed PCNA-dependent DNA chain elongation. The importance and roles of RFC and PCNA in M. thermoautotrophicum Delta H replication are discussed.

* This work was supported by National Institutes of Health Grant GM 38559 (to J. H.).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.

Dagger Supported by a Helen Hay Whitney post-doctoral fellowship. To whom correspondence should be addressed: Dept. of Molecular Biology, Memorial Sloan-Kettering Cancer Center, Box 97, 1275 York Ave., New York, NY 10021. Tel.: (212) 639-5895; Fax: (212) 717-3627; E-mail: z-kelman@ski.mskcc.org.

§ Professor of the American Cancer Society.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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