Werner syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability. The WS gene encodes a protein with helicase and exonuclease activities. Our previous studies indicated that the Werner syndrome protein (WRN) interacts with Ku, a heterodimeric factor of 70-and 80-kDa subunits implicated in the repair of double strand DNA breaks. Moreover, we demonstrated that Ku70/80 strongly stimulates and alters WRN exonuclease activity. In this report, we investigate further the association between WRN and Ku70/ 80. First, using various WRN deletion mutants we show that 50 amino acids at the amino terminus are required and sufficient to interact with Ku70/80. In addition, our data indicate that the region of Ku80 between amino acids 215 and 276 is necessary for binding to WRN. Then, we show that the amino-terminal region of WRN from amino acid 1 to 388, which comprise the exonuclease domain, can be efficiently stimulated by Ku to degrade DNA substrates, indicating that the helicase domain and the carboxyl-terminal tail are not required for the stimulatory process. Finally, using gel shift assays, we demonstrate that Ku recruits WRN to DNA. Taken together, these results suggest that Ku-mediated activation of WRN exonuclease activity may play an important role in a cellular pathway that requires processing of DNA ends.Werner syndrome (WS) 1 is a rare disorder characterized by the early appearance of many diseases characteristics of human aging such as atherosclerosis, osteoporosis and diabetes mellitus type II (1-3). In addition, WS patients are prone to many types of soft tissue tumors (4). Cells from WS patients display a shortened replicative life span and elevated levels of chromosomal abnormalities, including insertions, deletions and translocations (5, 6). The gene responsible for WS has been cloned and encodes a protein of 1,432 amino acids (WRN) (7). A nuclear localization signal has been identified at its carboxylterminal end (8), and all the described WS mutations result in a predicted truncated protein that fails to enter the nucleus. The central region of WRN is homologous to a seven-motif domain found in helicases from a wide variety of organisms, including bacteria (recQ), yeast (Sgs1), and human (Bloom syndrome (BLM), RecQL) (7, 9, 10). On the other hand, the amino-terminal region of WRN is quite unique among this family of helicases because it contains a sequence that resembles the exonuclease domain of Escherichia coli RNA polymerase I and RNase D (11, 12). Indeed, studies with the purified recombinant protein have shown that WRN displays both 3Ј-to 5Ј-exonuclease and 3Ј-to 5Ј-helicase activities (13-15).We have shown recently that Ku interacts with and alters the specificity of the WRN exonuclease (16). These results indicated that in the presence of Ku, WRN degradation of 3Ј-recessed strand of a partial DNA duplex is strongly stimulated. In addition, Ku alters the specificity of WRN so that the blunt end DNA duplex and 3Ј-protruding DNA...