Requirements for the nucleolytic processing of DNA ends by the Werner syndrome protein-Ku70/80 complex

Werner syndrome (WS) is an inherited disease characterized by premature ons et of aging, increased cancer incidence, and genomic instability. The WS ge ne 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 repai r of double strand DNA breaks, Moreover, we demonstrated that Ku70/80 stron gly stimulates and alters WRN exonuclease activity. In this report, we inve stigate further the association between WRN and Ku70/ 80. First, using vari ous 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 necess ary 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 ef ficiently stimulated by Ku to degrade DNA substrates, indicating that the h elicase domain and the carboxyl-terminal tail are not required for the stim ulatory process. Finally, using gel shift assays, we demonstrate that Ku re cruits WRN to DNA, Taken together, these results suggest that Ku-mediated a ctivation of WRN exonuclease activity may play an important role in a cellu lar pathway that requires processing of DNA ends.