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Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging. The gene responsible for the syndrome was recently cloned and shown to encode a protein with strong homology to DNA/RNA helicases. In addition, the Werner syndrome protein (WRN) possesses an exonuclease activity. Based on the homology to helicases it has been proposed that WRN functions in some aspects of DNA replication, recombination, or repair. However, there is currently no evidence of a role of WRN in any of these processes; therefore, its biological function remains unknown. Using a biochemical approach, we have identified two polypeptides that bind to the WRN protein. Peptide sequence analysis indicates that the two proteins are identical to Ku70 and Ku80, a heterodimer involved in double strand DNA break repair by non-homologous DNA end joining. Protein-protein interaction studies reveal that WRN binds directly to Ku80 and that this interaction is mediated by the amino terminus of WRN. In addition, we show that the binding of Ku alters the specificity of the WRN exonuclease. These results suggest a potential involvement of WRN in the repair of double strand DNA breaks.Werner syndrome (WS) 1 is a recessive autosomal disorder leading to premature aging (1). Individuals with WS develop accelerated atherosclerosis, osteoporosis, and a higher incidence of several types of tumors (2-4). Somatic cells of WS patients display a shortened replicative life span and elevated rates of chromosome translocation, rearrangements, and deletions (5, 6). The gene defective in WS has been identified and encodes a protein that possesses both exonuclease and helicase activities (7-13). A nuclear localization signal has also been identified at its carboxyl-terminal end. All the WS mutations that have been identified result in a nonsense mutation or frameshift, leading to a predicted truncated protein. Thus, it is thought that the truncated protein fails to enter the nucleus and is subsequently degraded. Immunocytochemical studies have localized the Werner syndrome protein (WRN) in the nucleoli of actively growing human cells, whereas serum deprivation causes a relocalization to the nucleus (14,15). Despite all the available information, the cellular function of WRN remains unknown (16). To understand the molecular basis of WS, we searched for nuclear factors that associate with the WRN protein. Here we report that WRN interacts with the Ku70/80 heterodimer (Ku), a factor involved in the repair of DNA double strand breaks (DSBs) (17)(18)(19)(20). More importantly, our results show that the exonuclease activity of WRN on 3Ј-recessed DNA ends is strongly stimulated by Ku. Moreover, in the presence of Ku, WRN can efficiently process both bluntended DNA and the 3Ј-protruding strand of a partial duplex DNA. These results suggest that WRN, through direct physical interaction with the Ku70/80 heterodimer, may be involved in processing of DSBs, providing a link between DNA repair and aging. EXPERIMENTAL PROCEDURESProtein Binding Assay and Peptide ...

Werner's syndrome (WS) is an autosomal recessive disorder in humans characterized by the premature development of a partial array of age-associated pathologies. WRN, the gene defective in WS, encodes a 1432 amino acid protein (hWRN) with intrinsic 3'-->5' DNA helicase activity. We recently showed that hWRN is also a 3'-->5' exonuclease. Here, we further characterize the hWRN exonuclease. hWRN efficiently degraded the 3' recessed strands of double-stranded DNA or a DNA-RNA heteroduplex. It had little or no activity on blunt-ended DNA, DNA with a 3' protruding strand, or single-stranded DNA. The hWRN exonuclease efficiently removed a mismatched nucleotide at a 3' recessed terminus, and was capable of initiating DNA degradation from a 12-nt gap, or a nick. We further show that the mouse WRN (mWRN) is also a 3'-->5' exonuclease, with substrate specificity similar to that of hWRN. Finally, we show that hWRN forms a trimer and interacts with the proliferating cell nuclear antigen in vitro. These findings provide new data on the biochemical activities of WRN that may help elucidate its role(s) in DNA metabolism.

Werner's syndrome (WS) is an inherited disease characterized by genomic instability and premature aging. The WS gene encodes a protein (WRN) with helicase and exonuclease activities. We have previously reported that WRN interacts with Ku70/80 and this interaction strongly stimulates WRN exonuclease activity. To gain further insight on the function of WRN and its relationship with the Ku heterodimer, we established a cell line expressing tagged WRN H , a WRN point mutant lacking helicase activity, and used affinity purification, immunoblot analysis and mass spectroscopy to identify WRNassociated proteins. To this end, we identified three proteins that are stably associated with WRN in nuclear extracts. Two of these proteins, Ku70 and Ku80, were identified by immunoblot analysis. The third polypeptide, which was identified by mass spectrometry analysis, is identical to poly(ADP-ribose) polymerase-1(PARP-1), a 113-kDa enzyme that functions as a sensor of DNA damage. Biochemical fractionation studies and immunoprecipitation assays and studies confirmed that endogenous WRN is associated with subpopulations of PARP-1 and Ku70/80 in the cell. Protein interaction assays with purified proteins further indicated that PARP-1 binds directly to WRN and assembles in a complex with WRN and Ku70/80. In the presence of DNA and NAD ؉ , PARP-1 poly(ADP-ribosyl)ates itself and Ku70/80 but not WRN, and gel-shift assays showed that poly-(ADP-ribosyl)ation of Ku70/80 decreases the DNA-binding affinity of this factor. Significantly, (ADP-ribosyl)ation of Ku70/80 reduces the ability of this factor to stimulate WRN exonuclease, suggesting that covalent modification of Ku70/80 by PARP-1 may play a role in the regulation of the exonucleolytic activity of WRN.Werner's syndrome (WS) 1 is a human genetic disease with many features of premature aging (1, 2). The first signs of this disorder appear soon after puberty, with the symptoms becoming fully evident in individuals between 20 and 30 years old. Individuals with WS display a high incidence of diseases associated with normal aging, including atherosclerosis, osteoporosis, type II diabetes mellitus, and cancer. Myocardial infarction and cancer are the most common causes of death among WS patients. The median age of death is ϳ47 years (1, 3). Cells isolated from WS patients show genomic instability and a shorter replicative life span (4). The genomic instability is characterized by an elevated rate of chromosomal translocations and extensive genomic deletions (5). These findings suggest that genomic instability underlies the development of the diseases associated with WS. Cultured cells from WS patients are also hypersensitive to some DNA damaging agents (4), suggestive of a defect in the repair of specific DNA lesions.Werner's syndrome is caused by mutations within a single gene, which is located on chromosome 8 (6). The cDNA encodes a protein (Werner's syndrome protein, WRN) with strong homology to a class of enzymes called RecQ helicases (7). In addition, the amino-terminal region of WR...

Background and Purpose-Although recent trials have suggested that stenting is worse than medical therapy for patients with severe symptomatic intracranial atherosclerotic stenosis, it is not clear whether this conclusion applies to a subset of patients with hypoperfusion symptoms. To justify for a new trial in China, we performed a multicenter prospective registry study to evaluate the safety and efficacy of endovascular stenting within 30 days for patients with severe symptomatic intracranial atherosclerotic stenosis. Methods-Patients with symptomatic intracranial atherosclerotic stenosis caused by 70% to 99% stenosis combined with poor collaterals were enrolled. The patients were treated either with balloon-mounted stent or with balloon predilation plus self-expanding stent as determined by the operators following a guideline. The primary outcome within 30 days is stroke, transient ischemic attack, and death after stenting. The secondary outcome is successful revascularization. The baseline characteristics and outcomes of the 2 treatment groups were compared. Results-From September 2013 to January 2015, among 354 consecutive patients, 300 patients (aged 58.3±9.78 years) were recruited, including 159 patients treated with balloon-mounted stent and 141 patients with balloon plus self-expanding stent. The 30-day rate of stroke, transient ischemic attack, and death was 4.3%. Successful revascularization was 97.3%. Patients treated with balloon-mounted stent were older, less likely to have middle cerebral artery lesions, more likely to have vertebral artery lesions, more likely to have Mori A lesions, less likely to have Mori C lesions, and likely to have lower degree of residual stenosis than patients treated with balloon plus self-expanding stent. Conclusions-The short-term safety and efficacy of endovascular stenting for patients with severe symptomatic intracranial atherosclerotic stenosis in China is acceptable. Balloon-mounted stent may have lower degree of residual stenosis than self-expanding stent. Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01968122.