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M111446200v1
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Papers In Press, published online ahead of print April 15, 2002
J. Biol. Chem, 10.1074/jbc.M111446200
Submitted on November 30, 2001
Revised on April 5, 2002
Accepted on April 14, 2002

Biochemical characterization of the DNA substrate specificity of Werner syndrome helicase

Robert M. Brosh Jr, Juwaria Waheed, and Joshua A. Sommers

Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD 21224

Corresponding Author: BroshR{at}grc.nia.nih.gov

Werner Syndrome is a hereditary premature aging disorder characterized by genome instability. The WRN gene product is a helicase/exonuclease that presumably functions in DNA metabolism. To understand the DNA structures WRN acts upon in vivo, we examined its substrate preferences for unwinding. WRN unwound a 3’ ssDNA tailed duplex substrate with streptavidin bound to the end of the 3’ ssDNA tail, suggesting that WRN does not require a free DNA end to unwind the duplex; however, WRN was completely blocked by streptavidin bound to the 3’ ssDNA tail six nucleotides upstream of the single-stranded/double-stranded DNA junction. WRN efficiently unwound forked duplex with streptavidin bound just upstream of the junction, suggesting that WRN recognizes elements of fork structure to initiate unwinding. WRN unwound two important intermediates of replication/repair, a 5’ ssDNA flap substrate and a synthetic replication fork. WRN was able to translocate on the lagging strand of the synthetic replication fork to unwind duplex ahead of the fork. For the 5’ flap structure, WRN specifically displaced the 5’ flap oligonucleotide, suggesting a role of WRN in Okazaki fragment processing. The ability of WRN to target DNA replication/repair intermediates may be relevant to its role in genome stability maintenance.


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