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Human Molecular Genetics Advance Access originally published online on July 28, 2004
Human Molecular Genetics 2004 13(19):2247-2261; doi:10.1093/hmg/ddh234
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Human Molecular Genetics, Vol. 13, No. 19 © Oxford University Press 2004; all rights reserved

In vivo function of the conserved non-catalytic domain of Werner syndrome helicase in DNA replication

Sudha Sharma1,2, Joshua A. Sommers1 and Robert M. Brosh, Jr1,*

1Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA and 2Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India

Received May 7, 2004; Accepted July 13, 2004

Werner syndrome is a genetic disorder characterized by genomic instability, elevated recombination and replication defects. The WRN gene encodes a RecQ helicase whose function(s) in cellular DNA metabolism is not well understood. To investigate the role of WRN in replication, we examined its ability to rescue cellular phenotypes of a yeast dna2 mutant defective in a helicase–endonuclease that participates with flap endonuclease 1 (FEN-1) in Okazaki fragment processing. Genetic complementation studies indicate that human WRN rescues dna2-1 mutant phenotypes of growth, cell cycle arrest and sensitivity to the replication inhibitor hydroxyurea or DNA damaging agent methylmethane sulfonate. A conserved non-catalytic C-terminal domain of WRN was sufficient for genetic rescue of dna2-1 mutant phenotypes. WRN and yeast FEN-1 were reciprocally co-immunoprecipitated from extracts of transformed dna2-1 cells. A physical interaction between yeast FEN-1 and WRN is demonstrated by yeast FEN-1 affinity pull-down experiments using transformed dna2-1 cells extracts and by ELISA assays with purified recombinant proteins. Biochemical analyses demonstrate that the C-terminal domain of WRN or BLM stimulates FEN-1 cleavage of its proposed physiological substrates during replication. Collectively, the results suggest that the WRN–FEN-1 interaction is biologically important in DNA metabolism and are consistent with a role of the conserved non-catalytic domain of a human RecQ helicase in DNA replication intermediate processing.

* To whom correspondence should be addressed. Tel: +14105588578; Fax: +1 4105588157; Email: broshR{at}grc.nia.nih.gov


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