Werner Protein Protects Nonproliferating Cells from Oxidative DNA Damage

Abstract

Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Although roles for WRN protein in DNA replication, recombination, and telomere maintenance have been suggested, the pathology of rapidly dividing cells is not a feature of Werner syndrome. To identify cellular events that are specifically vulnerable to WRN deficiency, we used RNA interference (RNAi) to knockdown WRN or BLM (the RecQ helicase mutated in Bloom syndrome) expression in primary human fibroblasts. Withdrawal of WRN or BLM produced accelerated cellular senescence phenotype and DNA damage response in normal fibroblasts, as evidenced by induction of γH2AX and 53BP1 nuclear foci. After WRN depletion, the induction of these foci was seen most prominently in nondividing cells. Growth in physiological (3%) oxygen or in the presence of an antioxidant prevented the development of the DNA damage foci in WRN-depleted cells, whereas acute oxidative stress led to inefficient repair of the lesions. Furthermore, WRN RNAi-induced DNA damage was suppressed by overexpression of the telomere-binding protein TRF2. These conditions, however, did not prevent the DNA damage response in BLM-ablated cells, suggesting a distinct role for WRN in DNA homeostasis in vivo. Thus, manifestations of Werner syndrome may reflect an impaired ability of slowly dividing cells to limit oxidative DNA damage.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/.

ACKNOWLEDGMENTS

We are grateful for the 53BP1 antibody and the full-length TRF2 and DN-TRF2 cDNAs provided by T. Halazonetis and T. de Lange, respectively. We thank S. Potluri for excellent help in the experimen-tal work.

A.M.S. is supported by a Mentored Clinical Scientist Award (KO8-AG01053) from the NIA. S.M.W. was awarded by the Ellison Medical Foundation Senior Scholar Program in Aging.