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Nucleic Acids Research, 2000, Vol. 28, No. 14 2762-2770
© 2000 Oxford University Press

Selective blockage of the 3'->5' exonuclease activity of WRN protein by certain oxidative modifications and bulky lesions in DNA

Amrita Machwe, Ruth Ganunis, Vilhelm A. Bohr* and David K. Orren

Laboratory of Molecular Genetics, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA

Individuals with mutations in the WRN gene suffer from Werner syndrome, a disease with early onset of many characteristics of normal aging. The WRN protein (WRNp) functions in DNA metabolism, as the purified polypeptide has both 3'->5' helicase and 3'->5' exonuclease activities. In this study, we have further characterized WRNp exonuclease activity by examining its ability to degrade double-stranded DNA substrates containing abnormal and damaged nucleo­tides. In addition, we directly compared the 3'->5' WRNp exonuclease activity with that of exo­nuclease III and the Klenow fragment of DNA polymerase I. Our results indicate that the presence of certain abnormal bases (such as uracil and hypoxanthine) does not inhibit the exonuclease activity of WRNp, exo­nuclease III or Klenow, whereas other DNA modifications, including apurinic sites, 8-oxoguanine, 8-oxoadenine and cholesterol adducts, inhibit or block WRNp. The ability of damaged nucleo­tides to inhibit exonucleolytic digestion differs significantly between WRNp, exonuclease III and Klenow, indicating that each exonuclease has a distinct mechanism of action. In addition, normal and modified DNA substrates are degraded similarly by full-length WRNp and an N-terminal fragment of WRNp, indicating that the specificity for this activity lies mostly within this region. The biochemical and physiological significance of these results is discussed.

* To whom correspondence should be addressed. Tel: +1 410 558 8162; Fax: +1 410 558 8157; Email: vbohr@nih.gov Present addresses: Amrita Machwe and David K. Orren, Graduate Center for Toxicology, 306 Health Sciences Research Building, Chandler Medical Center, University of Kentucky, Lexington, KY 40536-0305, USA


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