Requirements for the Nucleolytic Processing of DNA Ends by the Werner Syndrome Protein-Ku70/80 Complex

Abstract

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...

WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repair

“…WRN exonuclease activity is suppressed by interaction with p53 (Brosh et al ., 2001) or BLM (von , and stimulated by interaction with Ku70/80 (Li & Comai, 2001) or phosphorylation . WRN helicase activity is stimulated by interaction with p53 (Yang et al, 2002), replication protein A (RPA) (Shen et al, 1998), telomere repeat binding factor 2 (TRF2) and phosphorylation .…”

Biochemical and Kinetic Characterization of the DNA Helicase and Exonuclease Activities of Werner Syndrome Protein

“…The low level of WRN exonuclease activity in the presence of Mg 2ϩ and ATP can be explained by the robust unwinding of the forked duplex DNA substrate by WRN (Fig. 5A, lane 2), resulting in intact ssDNA that is not efficiently acted upon by WRN exonuclease when only Mg 2ϩ and/or ATP is present (34,51). Collectively these results demonstrate that in contrast to Mg 2ϩ , Zn 2ϩ dramatically stimulated WRN exonuclease activity on the forked duplex DNA substrate in the presence of ATP but failed to serve as a cofactor for WRN-catalyzed DNA unwinding.…”

“…10D, lane 7), but the extent of degradation was not as great as that observed for N-WRN in the presence of 100 M Zn 2ϩ when ATP was absent. Although Mg 2ϩ can serve as a cofactor for N-WRN exonuclease activity, a greater concentration (4 or 5 mM) is required for significant exonuclease activity (41,51).…”

Deficient DNA repair in the human progeroid disorder, Werner syndrome

“…A search for protein interactions with the WRN C-terminal region identified the Ku heterodimer as the most prominent binder [8]. Ku stimulates the WRN 3 -5 exonuclease and increases its processivity [8,20,21]. Ku is part of the DNA-PK complex, and associates with DNA and the DNA-PK catalytic subunit to form an active kinase [19].…”