Biochemical Characterization of the DNA Substrate Specificity of Werner Syndrome Helicase

Werner syndrome is a hereditary premature aging disorder characterized by genome instability. The product of the gene defective in WS, WRN, 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′-single-stranded (ss)DNA-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 6 nucleotides upstream of the single-stranded/double-stranded DNA junction. WRN efficiently unwound the forked duplex with streptavidin bound just upstream of the junction, suggesting that WRN recognizes elements of the 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. Werner syndrome is a hereditary premature aging disorder characterized by genome instability. The product of the gene defective in WS, WRN, 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′-single-stranded (ss)DNA-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 6 nucleotides upstream of the single-stranded/double-stranded DNA junction. WRN efficiently unwound the forked duplex with streptavidin bound just upstream of the junction, suggesting that WRN recognizes elements of the 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. Werner syndrome adenosine 5′-3-O-(thio)triphosphate double-stranded flap endonuclease 1 single-stranded Werner syndrome (WS)1 is an autosomal recessive disease that displays symptoms of premature aging after adolescence (1Martin G.M. Birth Defects Orig. Artic. Ser. 1978; 14: 5-39PubMed Google Scholar). WS is characterized by the early onset of age-related symptoms including gray hair, wrinkled skin, cataracts, atherosclerosis, diabetes, osteoporosis, and cancer. WS cells grown in culture display marked chromosomal instability characterized by elevated rates of chromosomal translocations and rearrangements (2Salk D., Au, K. Hoehn H. Martin G.M. Cytogenet. Cell Genet. 1981; 30: 92-107Crossref PubMed Scopus (212) Google Scholar, 3Salk D. Bryant E. Hoehn H. Johnston P. Martin G.M. Adv. Exp. Med. 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PubMed Scopus Google Scholar). a substrate with to of a was that WRN with a with to the strand that is to Res. PubMed Scopus Google Scholar). WRN is a Res. PubMed Scopus Google Scholar, B. Genet. PubMed Scopus Google Scholar, M. Biol. PubMed Scopus Google Scholar), with the of to the of DNA and in the M.J. A. Res. PubMed Scopus Google Scholar). The of WRN that the an important role in a of genome of DNA is defective in WS is not well The of and in and characterized by genomic instability, premature and that the is to be important in the of disease P. Hum. Genet. 2001; PubMed Scopus Google Schol