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J. Biol. Chem., Vol. 280, Issue 33, 29494-29505, August 19, 2005

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Physical and Functional Mapping of the Replication Protein A Interaction Domain of the Werner and Bloom Syndrome Helicases^*

Kevin M. Doherty, Joshua A. Sommers, Matthew D. Gray, Jae Wan Lee, Cayetano von Kobbe¶, Nicolas H. Thoma||, Raichal P. Kureekattil**, Mark K. Kenny**, and Robert M. Brosh, Jr.

From the Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, Maryland 21224, the MDG Associates, Seattle, Washington 98126, the ^||Structural Biology Program, Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, and the ^**Department of Emergency Medicine, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York 10467

The single-stranded DNA-binding protein replication protein A (RPA) interacts with several human RecQ DNA helicases that have important roles in maintaining genomic stability; however, the mechanism for RPA stimulation of DNA unwinding is not well understood. To map regions of Werner syndrome helicase (WRN) that interact with RPA, yeast two-hybrid studies, WRN affinity pull-down experiments and enzyme-linked immunosorbent assays with purified recombinant WRN protein fragments were performed. The results indicated that WRN has two RPA binding sites, a high affinity N-terminal site, and a lower affinity C-terminal site. Based on results from mapping studies, we sought to determine if the WRN N-terminal region harboring the high affinity RPA interaction site was important for RPA stimulation of WRN helicase activity. To accomplish this, we tested a catalytically active WRN helicase domain fragment (WRN_H-R) that lacked the N-terminal RPA interaction site for its ability to unwind long DNA duplex substrates, which the wild-type enzyme can efficiently unwind only in the presence of RPA. WRN_H-R helicase activity was significantly reduced on RPA-dependent partial duplex substrates compared with full-length WRN despite the presence of RPA. These results clearly demonstrate that, although WRN_H-R had comparable helicase activity to full-length WRN on short duplex substrates, its ability to unwind RPA-dependent WRN helicase substrates was significantly impaired. Similarly, a Bloom syndrome helicase (BLM) domain fragment, BLM_642–1290, that lacked its N-terminal RPA interaction site also unwound short DNA duplex substrates similar to wild-type BLM, but was severely compromised in its ability to unwind long DNA substrates that full-length BLM helicase could unwind in the presence of RPA. These results suggest that the physical interaction between RPA and WRN or BLM helicases plays an important role in the mechanism for RPA stimulation of helicase-catalyzed DNA unwinding.

Received for publication, January 18, 2005 , and in revised form, June 10, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

^¶ Current address: Centro Nacional de Investigaciones Oncologicas (CNIO), Telomeres and Telomerase Group, Madrid 28029, Spain.

To whom correspondence should be addressed: Laboratory of Molecular Gerontology, NIA, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224. Tel.: 410-558-8578; Fax: 410-558-8157; E-mail: BroshR{at}grc.nia.nih.gov.

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