Central Role for the Werner Syndrome Protein/Poly(ADP-Ribose) Polymerase 1 Complex in the Poly(ADP-Ribosyl)ation Pathway after DNA Damage

Abstract

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...

A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification

“…To date there is no evidence that WRN interacts with XRCC1 although it does form a complex with PARP1 [86,87]. WRN is classified as a RecQ family DNA helicase but has the unique feature of containing a 3'-5' exonuclease function.…”

“…Specifically, WRN exonuclease activity can act in conjunction with pol ß, a protein with no intrinsic 3'-5' proofreading activity [89]. WRN also partners with PARP1, the DNA singlestrand nick sensing protein [87].…”

Poly(ADP-ribose) Polymerase 1 (PARP-1) Binds to 8-Oxoguanine-DNA Glycosylase (OGG1)

“…We used PCR to clone the DNA binding domain (1-373 amino acids) of PARP-1 into the pET28a His vector using PARP-1 cDNA as a template (39). We used a standard purification procedure from Qiagen that included a thrombin cleavage step to purify the protein to homogeneity.…”

Poly(ADP-ribose) polymerase-1 polymorphisms, expression and activity in selected human tumour cell lines

“…Poly(ADP-ribose) polymerase-1 is activated 100-fold by DNA strand breaks and has a major role in DNA singlestrand break repair (which is part of base excision repair) (Dantzer et al, 1999(Dantzer et al, , 2000. Recent data also suggest that PARP-1 is involved in DNA double-strand break (DSB) repair (von Kobbe et al, 2003); however, its role in DSB repair remains unclear. As a result of compromised repair, PARP-1 deficient or inhibited cells are more sensitive to DNA-damaging agents (g-irradiation, topoisomerase inhibitors, alkylating agents) (Curtin, 2005).…”