Functional Interaction between Ku and the Werner Syndrome Protein in DNA End Processing

Abstract

Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging. The gene responsible for the syndrome was recently cloned and shown to encode a protein with strong homology to DNA/RNA helicases. In addition, the Werner syndrome protein (WRN) possesses an exonuclease activity. Based on the homology to helicases it has been proposed that WRN functions in some aspects of DNA replication, recombination, or repair. However, there is currently no evidence of a role of WRN in any of these processes; therefore, its biological function remains unknown. Using a biochemical approach, we have identified two polypeptides that bind to the WRN protein. Peptide sequence analysis indicates that the two proteins are identical to Ku70 and Ku80, a heterodimer involved in double strand DNA break repair by non-homologous DNA end joining. Protein-protein interaction studies reveal that WRN binds directly to Ku80 and that this interaction is mediated by the amino terminus of WRN. In addition, we show that the binding of Ku alters the specificity of the WRN exonuclease. These results suggest a potential involvement of WRN in the repair of double strand DNA breaks.Werner syndrome (WS) 1 is a recessive autosomal disorder leading to premature aging (1). Individuals with WS develop accelerated atherosclerosis, osteoporosis, and a higher incidence of several types of tumors (2-4). Somatic cells of WS patients display a shortened replicative life span and elevated rates of chromosome translocation, rearrangements, and deletions (5, 6). The gene defective in WS has been identified and encodes a protein that possesses both exonuclease and helicase activities (7-13). A nuclear localization signal has also been identified at its carboxyl-terminal end. All the WS mutations that have been identified result in a nonsense mutation or frameshift, leading to a predicted truncated protein. Thus, it is thought that the truncated protein fails to enter the nucleus and is subsequently degraded. Immunocytochemical studies have localized the Werner syndrome protein (WRN) in the nucleoli of actively growing human cells, whereas serum deprivation causes a relocalization to the nucleus (14,15). Despite all the available information, the cellular function of WRN remains unknown (16). To understand the molecular basis of WS, we searched for nuclear factors that associate with the WRN protein. Here we report that WRN interacts with the Ku70/80 heterodimer (Ku), a factor involved in the repair of DNA double strand breaks (DSBs) (17)(18)(19)(20). More importantly, our results show that the exonuclease activity of WRN on 3Ј-recessed DNA ends is strongly stimulated by Ku. Moreover, in the presence of Ku, WRN can efficiently process both bluntended DNA and the 3Ј-protruding strand of a partial duplex DNA. These results suggest that WRN, through direct physical interaction with the Ku70/80 heterodimer, may be involved in processing of DSBs, providing a link between DNA repair and aging. EXPERIMENTAL PROCEDURESProtein Binding Assay and Peptide ...

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