Functional and Physical Interaction between WRN Helicase and Human Replication Protein A

doi:10.1074/jbc.274.26.18341

Functional and Physical Interaction between WRN Helicase and Human Replication Protein A*

From the ^‡Laboratory of Molecular Genetics, NIA, National Institutes of Health, Baltimore, Maryland 21224,^§Aarhus University, Forskerparken, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark, and the ^¶Montefiore Medical Center, The Albert Einstein Cancer Center, Bronx, New York 10467

Abstract

The human premature aging disorder Werner syndrome (WS) is associated with a large number of symptoms displayed in normal aging. The WRN gene product, a DNA helicase, has been previously shown to unwind short DNA duplexes (≤53 base pairs) in a reaction stimulated by single-stranded DNA-binding proteins. We have studied the helicase activity of purified WRN protein on a variety of DNA duplex substrates to characterize the unwinding properties of the enzyme in greater detail. WRN helicase can catalyze unwinding of long duplex DNA substrates up to 849 base pairs in a reaction dependent on human replication protein A (hRPA). Escherichia coli SSB and bacteriophage T4 gene 32 protein (gp32) completely failed to stimulate WRN helicase to unwind long DNA duplexes indicating a specific functional interaction between WRN and hRPA. So far, there have been no reports of any physical interactions between WRN helicase and other proteins. In support of the functional interaction, we demonstrate a direct interaction between WRN and hRPA by coimmunoprecipitation of purified proteins. The physical and functional interaction between WRN and hRPA suggests that the two proteins may function together in vivo in a pathway of DNA metabolism such as replication, recombination, or repair.

Footnotes

↵* 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.
↵2 Balajee, A. S., Machwe, A., May, A., Gray, M. D., Oshima, J., Martin, G. M., Nehlin, J. O., Brosh, R. M., Jr., Orren, D. K., and Bohr, V. A., Mol. Biol. Cell, in press.
↵3 D. K. Orren, R. M. Brosh, Jr., J. O. Nehlin, A. Machwe, M. D. Gray, and V. A. Bohr, submitted for publication.
↵4 P. Ravn, unpublished data.
Abbreviations:

WS

Werner syndrome

SSB

single-stranded DNA binding protein

bp

base pairs

hRPA

human replication protein A

ESSB

E. coli SSB

dsDNA

double-stranded DNA

ssDNA

single-stranded DNA

PMSF

phenylmethylsulfonyl fluoride

BSA

bovine serum albumin

PIPES

1,4-piperazinediethanesulfonic acid

FFA1

focus-forming activity 1

scFv

single chain antibody fragments
- Received February 22, 1999.
- Revision received April 12, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.