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Human Molecular Genetics Advance Access published online on July 28, 2004
Human Molecular Genetics, doi:10.1093/hmg/ddh234
© 2004 by Oxford University Press
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Article

In vivo function of the conserved non-catalytic domain of werner syndrome helicase in dna replication

Sudha Sharma 1, Joshua A. Sommers 2, Robert M. Brosh Jr.2*

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

* To whom correspondence should be addressed. E-mail: broshR{at}grc.nia.nih.gov.


  Abstract

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 are not well understood. To investigate a 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 methyl methane 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.


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