Werner syndrome (WS) is characterized by features of premature aging and is caused by loss of the RecQ helicase protein WRN. WS fibroblasts display defects associated with telomere dysfunction, including accelerated telomere erosion and premature senescence. In yeast, RecQ helicases act in an alternative pathway for telomere lengthening (ALT) via homologous recombination. We found that WRN associates with telomeres when dissociation of telomeric D loops is likely during replication and recombination. In human ALT cells, WRN associates directly with telomeric DNA. The majority of TRF1/PCNA colocalizing foci contained WRN in live S phase ALT cells but not in telomerase-positive HeLa cells. Biochemically, the WRN helicase and 3' to 5' exonuclease act simultaneously and cooperate to release the 3' invading tail from a telomeric D loop in vitro. The telomere binding proteins TRF1 and TRF2 limit digestion by WRN. We propose roles for WRN in dissociating telomeric structures in telomerase-deficient cells.

Genome instability is a characteristic of cancer and aging, and is a hallmark of the premature aging disorder Werner syndrome (WS). Evidence suggests that the Werner syndrome protein (WRN) contributes to the maintenance of genome integrity through its involvement in DNA repair. In particular, biochemical evidence indicates a role for WRN in base excision repair (BER). We have previously reported that WRN helicase activity stimulates DNA polymerase beta (pol β) strand displacement synthesis in vitro. In this report we demonstrate that WRN exonuclease activity can act cooperatively with pol β, a polymerase lacking 3′–5′ proofreading activity. Furthermore, using small interference RNA technology, we demonstrate that WRN knockdown cells are hypersensitive to the alkylating agent methyl methanesulfonate, which creates DNA damage that is primarily repaired by the BER pathway. In addition, repair assays using whole cell extracts from WRN knockdown cells indicate a defect in long patch (LP) BER. These findings demonstrate that WRN plays a direct role in the repair of methylation-induced DNA damage, and suggest a role for both WRN helicase and exonuclease activities together with pol β during LP BER.

A defect in the Werner syndrome protein (WRN) leads to the premature aging disease Werner syndrome (WS). Hallmark features of cells derived from WS patients include genomic instability and hypersensitivity to certain DNA-damaging agents. WRN contains a highly conserved region, the RecQ conserved domain, that plays a central role in protein interactions. We searched for proteins that bound to this region, and the most prominent direct interaction was with poly(ADP-ribose) polymerase 1 (PARP-1), a nuclear enzyme that protects the genome by responding to DNA damage and facilitating DNA repair. In pursuit of a functional interaction between WRN and PARP-1, we found that WS cells are deficient in the poly(ADP-ribosyl)ation pathway after they are treated with the DNA-damaging agents H 2 O 2 and methyl methanesulfonate. After cellular stress, PARP-1 itself becomes activated, but the poly(ADP-ribosyl)ation of other cellular proteins is severely impaired in WS cells. Overexpression of the PARP-1 binding domain of WRN strongly inhibits the poly(ADP-ribosyl)ation activity in H 2 O 2 -treated control cell lines. These results indicate that the WRN/PARP-1 complex plays a key role in the cellular response to oxidative stress and alkylating agents, suggesting a role for these proteins in the base excision DNA repair pathway.Werner syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging and the early onset of cancer (26). WS is caused by mutations in the gene (WRN) encoding the Werner syndrome protein (WRN) (51). WRN is a multifunctional protein which possesses three catalytic activities, which reside in the amino terminus (3Ј-5Ј exonuclease) and the central part of the protein (DNA-dependent ATPase and 3Ј-5Ј helicase) (5). The RecQ conserved (RQC) motif resides in the C-terminal part of WRN and is present in almost all of the RecQ family members (51). Recently, we have shown that a region of 144 amino acids (aa) of the RQC domain binds to and stimulates flap endonuclease 1 (FEN-1) (7), binds to the Bloom's syndrome protein (49) and telomere repeat binding factor 2 (30), and contains a nuclear localization signal-dependent nucleolar targeting sequence (48). Thus, the highly conserved RQC domain of WRN appears to play a very important role in mediating WRN protein interactions and in regulating the nuclear trafficking (nucleolar targeting) of the protein. In an effort to better understand the functional role(s) of this domain, we performed a series of pull-down experiments to identify proteins that specifically bind to the WRN RQC domain. The most prominent binder that we identified was poly-(ADP-ribose) polymerase 1 (PARP-1), whose binding represents a novel protein interaction with WRN.PARP-1 is a nuclear enzyme belonging to the DNA damage surveillance network. The protein responds to DNA damage by transferring 50 to 200 molecules of ADP-ribose to various nuclear proteins, including transcription factors, histones, and PARP-1 itself (8). This poly(ADP-ribosyl)ation activity of PARP-1 appears to be...