Central role for the Werner syndrome protein/poly(ADP-ribose) polymerase 1 complex in the poly(ADP-ribosyl)ation pathway after DNA damage.

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 (3′-5′ exonuclease) and the central part of the protein (DNA-dependent ATPase and 3′-5′ 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 important for maintaining genomic integrity (47), and it has been associated with longevity (8, 16, 52). Furthermore, PARP-1 is activated by several DNA-damaging agents, such as hydrogen peroxide (H2O2), alkylating agents (including methyl methanesulfonate [MMS]), bleomycin, and radiation. Our finding in the present study that PARP-1 is the most prominent protein associated with the WRN RQC domain prompted further analysis of this protein interaction and an investigation of the biological significance of this interaction. We report here that WS cells are deficient in the poly(ADP-ribosyl)ation pathway after H2O2 and MMS treatment. After DNA damage, PARP-1 becomes auto-poly(ADP-ribosyl)ated in WS cells, but the poly(ADP-ribosyl)ation of other cellular proteins in WS cells is undetectable by immunofluorescence and Western blot analysis. The WRN/PARP-1 interaction appears to depend on the poly(ADP-ribosyl)ation state of PARP-1. In further support of a biological function of the WRN/PARP-1 interaction, we show that overexpression of the PARP-1 binding domain of WRN strongly inhibits the poly(ADP-ribosyl)ation activity in H2O2-treated control cell lines. Altogether, these results indicate that the WRN/PARP-1 complex plays a key role in the cellular response to oxidative stress and DNA alkylation damage. The DNA damage caused by these reagents is generally repaired by the base excision repair (BER) machinery. A role for the WRN/PARP-1 complex in BER is discussed.