Deletion Analysis of the Large Subunit p140 in Human Replication Factor C Reveals Regions Required for Complex Formation and Replication Activities*
- From the Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center and
- § Microbiology Department and Howard Hughes Medical Institute, Cornell University Medical College, New York, New York 10021
- ↵¶ Professor of the American Cancer Society. To whom correspondence should be addressed: Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 97, New York, NY 10021.
Abstract
Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are processivity factors for eukaryotic DNA polymerases δ and ϵ. RFC contains multiple activities, including its ability to recognize and bind to a DNA primer end and load the ring-shaped PCNA onto DNA in an ATP-dependent reaction. PCNA then tethers the polymerase to the template allowing processive DNA chain elongation. Human RFC consists of five distinct subunits (p140, p40, p38, p37, and p36), and RFC activity can be reconstituted from the five cloned gene products. To characterize the role of the large subunit p140 in the function of the RFC complex, deletion mutants were created that defined a region within the p140 C terminus required for complex formation with the four small subunits. Deletion of the p140 N-terminal half, including the DNA ligase homology domain, resulted in the formation of an RFC complex with enhanced activity in replication and PCNA loading. Deletion of additional N-terminal amino acids, including those constituting the RFC homology box II that is conserved among all five RFC subunits, disrupted RFC replication function. DNA primer end recognition and PCNA binding activities, located in the p140 C-terminal half, were unaffected in this mutant, but PCNA loading was abolished.
Footnotes
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↵‡ Enrolled in the graduate program at the Physiologisch-chemisches Institut, Universität Tübingen, and supported by the German Academic Exchange Service (DAAD) through funds of the Zweites Hochschulsonderprogramm.
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↵* These studies were supported by National Institutes of Health Grants GM 38559 (to J. H.) and GM 38839 (to M. O'D.). 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.
- Received December 12, 1996.
- © 1997 by The American Society for Biochemistry and Molecular Biology, Inc.
