p53 Modulates the exonuclease activity of Werner syndrome protein.

Werner syndrome (WS) is characterized by the early onset of symptoms of premature aging, cancer, and genomic instability. The molecular basis of the defects is not understood but presumably relates to the DNA helicase and exonuclease activities of the protein encoded by the WRN gene that is mutated in the disease. The attenuation of p53-mediated apoptosis in WS cells and reported physical interaction between WRN and the tumor suppressor p53 suggest that p53 and WRN functionally interact in a pathway necessary for the normal cellular response. In this study, we have demonstrated that p53 inhibits the exonuclease activity of the purified full-length recombinant WRN protein. p53 did not have an effect on a truncated amino-terminal WRN fragment that retains exonuclease activity but lacks the physical interaction domain for p53 located in the carboxyl terminus. Two naturally occurring p53 mutants found in human cancer displayed a reduced ability to inhibit WRN exonuclease activity. In cells arrested in S phase with hydroxyurea, WRN exits the nucleolus and colocalizes with p53 in the nucleoplasm. The regulation of WRN function by p53 is likely to play an important role in the maintenance of genomic integrity and prevention of cancer and other clinical symptoms associated with WS.     

Identification and characterization of a Drosophila ortholog of WRN exonuclease that is required to maintain genome integrity

The premature human aging Werner syndrome (WS) is caused by mutation of the RecQ-family WRN helicase, which is unique in possessing also 3'-5' exonuclease activity. WS patients show significant genomic instability with elevated cancer incidence. WRN is implicated in restraining illegitimate recombination, especially during DNA replication. Here we identify a Drosophila ortholog of the WRN exonuclease encoded by the CG7670 locus. The predicted DmWRNexo protein shows conservation of structural motifs and key catalytic residues with human WRN exonuclease, but entirely lacks a helicase domain. Insertion of a piggyBac element into the 5' UTR of CG7670 severely reduces gene expression. DmWRNexo mutant flies homozygous for this insertional allele of CG7670 are thus severely hypomorphic; although adults show no gross morphological abnormalities, females are sterile. Like human WS cells, we show that the DmWRNexo mutant flies are hypersensitive to the topoisomerase I inhibitor camptothecin. Furthermore, these mutant flies show highly elevated rates of mitotic DNA recombination resulting from excessive reciprocal exchange. This study identifies a novel WRN ortholog in flies and demonstrates an important role for WRN exonuclease in maintaining genome stability.     

Mutations in the WRN gene in mice accelerate mortality in a p53-null background

Werner's syndrome (WS) is a human disease with manifestations resembling premature aging. The gene defective in WS, WRN, encodes a DNA helicase. Here, we describe the generation of mice bearing a mutation that eliminates expression of the C terminus of the helicase domain of the WRN protein. Mutant mice are born at the expected Mendelian frequency and do not show any overt histological signs of accelerated senescence. These mice are capable of living beyond 2 years of age. Cells from these animals do not show elevated susceptibility to the genotoxins camptothecin or 4-NQO. However, mutant fibroblasts senesce approximately one passage earlier than controls. Importantly, WRN(-/-);p53(-/-) mice show an increased mortality rate relative to WRN(+/-);p53(-/-) animals. We consider possible models for the synergy between p53 and WRN mutations for the determination of life span.     

Comparison of proliferation and genomic instability responses to WRN silencing in hematopoietic HL60 and TK6 cells

Background

Werner syndrome (WS) results from defects in the RecQ helicase (WRN) and is characterized by premature aging and accelerated tumorigenesis. Contradictorily, WRN deficient human fibroblasts derived from WS patients show a characteristically slower cell proliferation rate, as do primary fibroblasts and human cancer cell lines with WRN depletion. Previous studies reported that WRN silencing in combination with deficiency in other genes led to significantly accelerated cellular proliferation and tumorigenesis. The aim of the present study was to examine the effects of silencing WRN in p53 deficient HL60 and p53 wild-type TK6 hematopoietic cells, in order to further the understanding of WRN-associated tumorigenesis.

Methodology/Principal Findings

We found that silencing WRN accelerated the proliferation of HL60 cells and decreased the cell growth rate of TK6 cells. Loss of WRN increased DNA damage in both cell types as measured by COMET assay, but elicited different responses in each cell line. In HL60 cells, but not in TK6 cells, the loss of WRN led to significant increases in levels of phosphorylated RB and numbers of cells progressing from G1 phase to S phase as shown by cell cycle analysis. Moreover, WRN depletion in HL60 cells led to the hyper-activation of homologous recombination repair via up-regulation of RAD51 and BLM protein levels. This resulted in DNA damage disrepair, apparent by the increased frequencies of both spontaneous and chemically induced structural chromosomal aberrations and sister chromatid exchanges.

Conclusions/Significance

Together, our data suggest that the effects of WRN silencing on cell proliferation and genomic instability are modulated probably by other genetic factors, including p53, which might play a role in the carcinogenesis induced by WRN deficiency.     

DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possesses both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.     

Distinct functions for WRN and TP53 in a shared pathway of cellular response to 1-beta-D-arabinofuranosylcytosine and bleomycin

Mutations in the WRN or the TP53 genes lead to spontaneous genetic instability, an elevated risk of tumor formation, and sensitivity to compounds that interfere with DNA replication, such as camptothecin and DNA interstrand cross-linking drugs. We investigated the hypothesis that WRN and TP53 are involved in cellular responses to DNA replication-blocking lesions by exposing WRN deficient and TP53 mutant lymphoblastoid cell lines (LCLs) to 1-beta-d-arabinofuranosylcytosine (AraC) and bleomycin. Loss of WRN or TP53 function resulted in induction of apoptosis and lesser proliferative survival in response to AraC and bleomycin. WRN and TP53 operate in a shared DNA damage response pathway, since in cells in which TP53 was inactivated by SV-40 transformation, no difference in AraC and bleomycin sensitivity was found regardless of WRN status. In contrast to TP53 mutant LCLs, WRN-deficient cells showed unaffected cell cycle arrest after AraC and bleomycin exposure, which indicates that WRN is not involved in DNA damage-activated cell cycle arrest. Neither WRN nor TP53 deficiency affected cellular recovery from exposure to AraC and bleomycin, which disagrees with a direct role in repair of these DNA lesions. Our results indicate that WRN and TP53 perform different functions in a shared DNA damage response pathway.     

Requirements for the nucleolytic processing of DNA ends by the Werner syndrome protein-Ku70/80 complex

Werner syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability. The WS gene encodes a protein with helicase and exonuclease activities. Our previous studies indicated that the Werner syndrome protein (WRN) interacts with Ku, a heterodimeric factor of 70- and 80-kDa subunits implicated in the repair of double strand DNA breaks. Moreover, we demonstrated that Ku70/80 strongly stimulates and alters WRN exonuclease activity. In this report, we investigate further the association between WRN and Ku70/80. First, using various WRN deletion mutants we show that 50 amino acids at the amino terminus are required and sufficient to interact with Ku70/80. In addition, our data indicate that the region of Ku80 between amino acids 215 and 276 is necessary for binding to WRN. Then, we show that the amino-terminal region of WRN from amino acid 1 to 388, which comprise the exonuclease domain, can be efficiently stimulated by Ku to degrade DNA substrates, indicating that the helicase domain and the carboxyl-terminal tail are not required for the stimulatory process. Finally, using gel shift assays, we demonstrate that Ku recruits WRN to DNA. Taken together, these results suggest that Ku-mediated activation of WRN exonuclease activity may play an important role in a cellular pathway that requires processing of DNA ends.     

The Werner syndrome gene: the molecular basis of RecQ helicase-deficiency diseases

Werner syndrome (WS) is an autosomal recessive genetic disorder that is manifested by genetic instability and premature onset of age-related diseases, including atherosclerosis and cancer. The gene that is mutated in WS cells (WRN) has been identified recently. Characterizations of the WRN gene product indicate that WRN encodes both a 3'-->5' DNA helicase, belonging to the Escherichiacoli RecQ helicase family, and a 3'-->5' DNA exonuclease. Studies to define the molecular mechanism of WRN-DNA transactions are currently underway in many laboratories. Preliminary results indicate that WRN functions as a key factor in resolving aberrant DNA structures that arise from DNA metabolic processes such as replication, recombination and/or repair, to preserve the genetic integrity in cells.     

Interactions between the Werner syndrome helicase and DNA polymerase delta specifically facilitate copying of tetraplex and hairpin structures of the d(CGG)n trinucleotide repeat sequence

Werner syndrome (WS) is an inherited disorder characterized by premature aging and genomic instability. The protein encoded by the WS gene, WRN, possesses intrinsic 3' --> 5' DNA helicase and 3' --> 5' DNA exonuclease activities. WRN helicase resolves alternate DNA structures including tetraplex and triplex DNA, and Holliday junctions. Thus, one function of WRN may be to unwind secondary structures that impede cellular DNA transactions. We report here that hairpin and G'2 bimolecular tetraplex structures of the fragile X expanded sequence, d(CGG)(n), effectively impede synthesis by three eukaryotic replicative DNA polymerases (pol): pol alpha, pol delta, and pol epsilon. The constraints imposed on pol delta-catalyzed synthesis are relieved, however, by WRN; WRN facilitates pol delta to traverse these template secondary structures to synthesize full-length DNA products. The alleviatory effect of WRN is limited to pol delta; neither pol alpha nor pol epsilon can traverse template d(CGG)(n) hairpin and tetraplex structures in the presence of WRN. Alleviation of pausing by pol delta is observed with Escherichia coli RecQ but not with UvrD helicase, suggesting a concerted action of RecQ helicases and pol delta. Our findings suggest a possible role of WRN in rescuing pol delta-mediated replication at forks stalled by unusual DNA secondary structures.     

WRN is required for ATM activation and the S-phase checkpoint in response to interstrand cross-link-induced DNA double-strand breaks

Werner syndrome (WS) is a human genetic disorder characterized by extensive clinical features of premature aging. Ataxia-telengiectasia (A-T) is a multisystem human genomic instability syndrome that includes premature aging in some of the patients. WRN and ATM, the proteins defective in WS and A-T, respectively, play significant roles in the maintenance of genomic stability and are involved in several DNA metabolic pathways. A role for WRN in DNA repair has been proposed; however, this study provides evidence that WRN is also involved in ATM pathway activation and in a S-phase checkpoint in cells exposed to DNA interstrand cross-link–induced double-strand breaks. Depletion of WRN in such cells by RNA interference results in an intra-S checkpoint defect, and interferes with activation of ATM as well as downstream phosphorylation of ATM target proteins. Treatment of cells under replication stress with the ATM kinase inhibitor KU 55933 results in a S-phase checkpoint defect similar to that observed in WRN shRNA cells. Moreover, γH2AX levels are higher in WRN shRNA cells than in control cells 6 and 16 h after exposure to psoralen DNA cross-links. These results suggest that WRN and ATM participate in a replication checkpoint response, in which WRN facilitates ATM activation in cells with psoralen DNA cross-link–induced collapsed replication forks.     

Rapamycin decreases DNA damage accumulation and enhances cell growth of WRN‐deficient human fibroblasts

Werner syndrome (WS), caused by mutations at the WRN helicase gene, is a progeroid syndrome characterized by multiple features consistent with accelerated aging. Aberrant double‐strand DNA damage repair leads to genomic instability and reduced replicative lifespan of somatic cells. We observed increased autophagy in WRN knockdown cells; this was further increased by short‐term rapamycin treatment. Long‐term rapamycin treatment resulted in improved growth rate, reduced accumulation of DNA damage foci and improved nuclear morphology; autophagy markers were reduced to near‐normal levels, possibly due to clearance of damaged proteins. These data suggest that protein aggregation plays a role in the development of WS phenotypes and that the mammalian target of rapamycin complex 1 pathway is a potential therapeutic target of WS.     

The enzymatic activities of the Werner syndrome protein are disabled by the amino acid polymorphism R834C

The Werner syndrome protein, WRN, is a member of the RecQ family of DNA helicases. It possesses both 3'-->5' DNA helicase and 3'-->5' DNA exonuclease activities. Mutations in WRN are causally associated with a rare, recessive disorder, Werner syndrome (WS), distinguished by premature aging and genomic instability; all are reported to result in loss of protein expression. In addition to WS-linked mutations, single nucleotide polymorphisms, with frequencies that exceed those of WS-associated mutations, are also present in WRN. We have initiated studies to determine if six of these polymorphisms affect the enzymatic activities of WRN. We show that two common polymorphisms, F1074L and C1367R, and two infrequent polymorphisms, Q724L and S1079L, exhibit little change in activity relative to wild-type WRN; the polymorphism, T172P, shows a small but consistent reduction of activity. However, an infrequent polymorphism, R834C, located in the helicase domain dramatically reduces WRN helicase and helicase-coupled exonuclease activity. The structure of the E. coli helicase core suggests that R834 may be involved in interactions with ATP. As predicted, substitution of Arg with Cys interferes with ATP hydrolysis that is absolutely required for unwinding DNA. R834C thus represents the first missense amino acid polymorphism in WRN that nearly abolishes enzymatic activity while leaving expression largely unaffected.