The Werner Syndrome Gene Product Co-purifies with the DNA Replication Complex and Interacts with PCNA and Topoisomerase I*
- From the ‡Department of Genetics, Harvard Medical School, the ‖Howard Hughes Medical Institute, Boston, Massachusetts 02115, and the ¶Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255
Abstract
Werner syndrome (WS) is a recessive disorder characterized by genomic instability and by the premature onset of a number of age-related diseases. To understand the molecular basis of this disease, we deleted a segment of the murine Wrn gene and created Wrn-deficient embryonic stem (ES) cells. At the molecular level, wild type—but not mutant—WS protein co-purifies through a series of centrifugation, chromatography, and sucrose gradient steps with the well characterized 17 S multiprotein DNA replication complex. Furthermore, wild type WS protein co-immunoprecipitates with a prominent component of the multiprotein replication complex, proliferating cell nuclear antigen (PCNA).In vitro studies also indicate that PCNA binds to a region in the N terminus portion of the WS protein containing a potential 3′-5′ exonuclease domain. Finally, human WS protein also co-immunoprecipitates with both PCNA and topoisomerase I. These results suggest that the WS protein interacts with several components of the DNA replication fork.
Footnotes
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↵* 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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↵§ Recipient of a Research Fellowship of the National Cancer Institute of Canada supported with funds provided by the Terry Fox Run.
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↵** To whom correspondence should be addressed: Dept. of Genetics, Harvard Medical School, Howard Hughes Medical Institute, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-7667; Fax: 617-432-7663.
- Abbreviations:
- WS
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Werner syndrome
- ES
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embryonic stem
- GST
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glutathione S-transferase
- PMSF
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phenylmethylsulfonyl fluoride
- DTT
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dithiothreitol
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- Received September 28, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
