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...

Summary Rothmund-Thomson syndrome (RTS) is an autosomal recessive hereditary disorder associated with mutation in RECQL4 gene, a member of the human RecQ helicases. The disease is characterized by genomic instability, skeletal abnormalities and predisposition to malignant tumors, especially osteosarcomas. The precise role of RECQL4 in cellular pathways is largely unknown, however recent evidence suggest its involvement in multiple DNA metabolic pathways. This study investigates the roles of RECQL4 in DNA double strand break (DSB) repair. The results show that RECQL4-deficient fibroblasts are moderately sensitive to γ-irradiation and accumulate more γH2AX and 53BP1 foci than control fibroblasts. This is suggestive of defects in efficient repair of DSB’s in the RECQL4 deficient fibroblasts. Real time imaging of live cells using laser confocal microscopy show that RECQL4 is recruited early to laser induced DSBs and remains for a shorter duration than WRN and BLM indicating its distinct role in repair of DSBs. Endogenous RECQL4 also colocalizes with γH2AX at the site of DSBs. The RECQL4 domain responsible for its DNA damage localization has been mapped to the unique N-terminus domain between amino acids 363–492, which shares no homology to recruitment domains of WRN and BLM to the DSBs. Further, the recruitment of RECQL4 to laser induced DNA damage is independent of functional WRN, BLM or ATM proteins. These results suggest distinct cellular dynamics for RECQL4 protein at the site of laser induced DSB and that it might play important roles in efficient repair of DSB’s.

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)-dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40-60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II-dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid-protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.

Werner syndrome (WS) is a premature aging disorder, displaying defects in DNA replication, recombination, repair, and transcription. It has been hypothesized that several WS phenotypes are secondary consequences of aberrant gene expression and that a transcription defect may be crucial to the development of the syndrome. We used cDNA microarrays to characterize the expression of 6,912 genes and ESTs across a panel of 15 primary human fibroblast cell lines derived from young donors, old donors, and WS patients. Of the analyzed genes, 6.3% displayed significant differences in expression when either WS or old donor cells were compared with young donor cells. This result demonstrates that the WS transcription defect is specific to certain genes. Transcription alterations in WS were strikingly similar to those in normal aging: 91% of annotated genes displayed similar expression changes in WS and in normal aging, 3% were unique to WS, and 6% were unique to normal aging. We propose that a defect in the transcription of the genes as identified in this study could produce many of the complex clinical features of WS. The remarkable similarity between WS and normal aging suggests that WS causes the acceleration of a normal aging mechanism. This finding supports the use of WS as an aging model and implies that the transcription alterations common to WS and normal aging represent general events in the aging process.W erner syndrome (WS) is an autosomal recessive disease characterized by early onset of many signs of normal aging, such as graying of the hair, scleroderma-like skin changes, ocular cataracts, diabetes, degenerative vascular disease, osteoporosis, and high incidence of some types of cancers (1). As a segmental progeroid syndrome, WS does not exhibit all of the features of normal aging but nevertheless is a very useful model system for the molecular study of normal aging.The molecular basis of WS is a single mutation in the WRN gene, resulting in a truncated WS protein (WRN) characterized by a loss of nuclear localization signal and protein function (2). WRN has been demonstrated to possess helicase and exonuclease activities (3, 4) and belongs to the RecQ family of helicases. Various defects in DNA replication, recombination, repair, and transcription are found in WS fibroblasts (reviewed in ref. 5). The mechanisms by which the biochemical deficiencies resulting from WRN mutations lead to the characteristic pathology of the syndrome are not yet understood. It has been hypothesized that several WS phenotypes are secondary consequences of aberrant gene expression (6) and that a transcription defect may be crucial to the development of the syndrome (7). Increasing evidence suggests that WRN has a role in transcription. Human WRN activates transcription in a yeast system (8), and recent studies from this laboratory demonstrated that RNA polymerase (pol) II transcription is reduced by 40-60% in WS cells, indicating a primary defect in transcription (7). Supporting this finding, we found that RNA pol II transcription i...

Cockayne syndrome (CS) is characterized by increased photosensitivity, growth retardation, and neurological and skeletal abnormalities. The recovery of RNA synthesis is abnormally delayed in CS cells after exposure to UV radiation. Gene-specific repair studies have shown a defect in the transcription-coupled repair (

SUMMARY The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6), promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB) repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.