Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51

BACKGROUND: Werner syndrome (WS) is an autosomal recessive disorder with many features of premature ageing. Cells derived from WS patients show genomic instability, aberrations in the S-phase and sensitivity to genotoxic agents. The gene responsible for WS (WRN) encodes a DNA helicase belonging to the RecQ helicase family. Although biochemical studies showed that the gene product of WRN (WRNp) interacts with proteins that participate in DNA metabolism, its precise biological function remains unclear. RESULTS: Using immunocytochemistry, we found that WRNp forms distinct nuclear foci in response to DNA damaging agents, including camptothecin (CPT), etoposide, 4-nitroquinolin-N-oxide and bleomycin. The presence of aphidicolin inhibited CPT-induced WRNp foci strongly but not bleomycin-induced foci. These WRNp foci overlapped with the foci of replication protein A (RPA) almost entirely and with the foci of Rad51 partially, implicating cooperative functions of these proteins in response to DNA damage. We also found that WRNp foci partially co-localize with sites of 5-bromo-2'-deoxy-uridine incorporation. CONCLUSIONS: These findings suggest that WRNp form nuclear foci in response to aberrant DNA structures, including DNA double-strand breaks and stalled replication forks. We propose that WRNp takes part in the homologous recombinational repair and in the processing of stalled replication forks.     

Pathways and functions of the Werner syndrome protein

Mutations in human WRN (also known as RECQ3) gene give rise to a rare autosomal recessive genetic disorder, Werner syndrome (WS). WS is a premature aging disease characterized by predisposition to cancer and early onset of symptoms related to normal aging including osteoporosis, ocular cataracts, graying and loss of hair, diabetes mellitus, arteriosclerosis, and atherosclerosis. This review focuses on the functional role of Werner protein (WRN) in guarding the genetic stability of cells, particularly by playing an integral role in the base excision repair, and at the telomere ends. Furthermore, in-depth biochemical investigations have significantly advanced our understanding of WRN protein regarding its binding partners and the site of protein-protein interaction. The mapping analysis of protein interaction sites in WRN for most of its binding partners have revealed a common site of protein-protein interaction in the RecQ conserved (RQC) region of WRN.     

The role of WRN in DNA repair is affected by post-translational modifications

Werner syndrome (WS) is an autosomal recessive progeroid disease characterized by genomic instability. WRN gene encodes one of the RecQ helicase family proteins, WRN, which has ATPase, helicase, exonuclease and single stranded DNA annealing activities. There is accumulating evidence suggesting that WRN contributes to the maintenance of genomic integrity through its involvement in DNA repair, replication and recombination. The role of WRN in these pathways can be modulated by its post-translational modifications in response to DNA damage. Here, we review the functional consequences of post-translational modifications on WRN as well as specific DNA repair pathways where WRN is involved and discuss how these modifications affect DNA repair pathways.     

Unwinding the molecular basis of the Werner syndrome

Werner syndrome (WS) is an autosomal recessive disease manifested by the premature onset of age-related phenotypes, including diseases such as atherosclerosis and cancer. This mimicry of normal aging with the possible exception of central nervous system manifestations has made it a focus of recent molecular studies on the pathophysiology of aging. In culture, cells obtained from patients with WS are genetically unstable, characterized by an increased frequency of nonclonal translocations and extensive DNA deletions. The WS gene product (WRN) is a DNA helicase belonging to the RecQ family, but is unique within this family in that it also contains an exonuclease activity. In addition to unwinding double-stranded DNA, WRN helicase is able to resolve aberrant DNA structures such as G4 tetraplexes, triplexes and 4-way junctions. Concordant with this structure-specificity, WRN exonuclease preferentially hydrolyzes alternative DNA that contains bubbles, extra-helical loops, 3-way junctions or 4-way junctions. WRN has been shown to bind to and/or functionally interact with other proteins, including replication protein A (RPA), proliferating cell nuclear antigen (PCNA), DNA topoisomerase I, Ku 86/70, DNA polymerase delta and p53. Each of these interacting proteins is involved in DNA transactions including those that resolve alternative DNA structures or repair DNA damage. The biochemical activities of WRN and the functions of WRN associated proteins suggest that in vivo WRN resolves DNA topological or structural aberrations that either occur during DNA metabolic processes such as recombination, replication and repair, or are the outcome of DNA damage.     

Characterisation of the interaction between WRN,the helicase/exonuclease defective in progeroid Werner's syndrome,and an essential replication factor,PCNA

Ageing is linked to the accumulation of replicatively senescent cells. The best model system to date for studying human cellular ageing is the progeroid Werner's syndrome (WS), caused by a defect in WRN, a recQ-like helicase that also possesses exonuclease activity. In this paper, we characterise the interaction between WRN and an essential replication factor, PCNA. We show that wild-type WRN protein physically associates with PCNA at physiological protein concentrations in normal cells, while no association is seen in cells from patients with WS. We demonstrate co-localisation of WRN and PCNA at replication factories, show that PCNA binds to two distinct functional sites on WRN, and suggest a mechanism by which association between WRN and PCNA may be regulated in cells on DNA damage and during DNA replication.     

Werner's syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle

Werner's syndrome (WS) is a rare autosomal recessive human disorder and the patients exhibit many symptoms of accelerated ageing in their early adulthood. The gene (WRN) responsible for WS has been biochemically characterised as a 3′–5′ helicase and is homologous to a number of RecQ superfamily of helicases. The yeast SGS1 helicase is considered as a human WRN homologue and SGS1 physically interacts with topoisomerases II and III. In view of this, it has been hypothesised that the WRN gene may also interact with topoisomerases II and III. The purpose of this study is to determine whether the loss of function of WRN protein alters the sensitivity of WS cells to agents that block the action of topoisomerase II. This study deals with the comparison of the chromosomal damage induced by the two anti-topoisomerase II drugs, VP-16 and amsacrine, in both G1 and G2 phases of the cell cycle, in lymphoblastoid cells from WS patients and from a healthy donor. Our results show that the WS cell lines are hypersensitive to chromosome damage induced by VP-16 and amsacrine only in the G2 phase of the cell cycle. No difference either in the yield of the induced aberrations or SCEs was found after treatment of cells at G1 stage. These data might suggest that in WS cells, because of the mutation of the WRN protein, the inhibition of topoisomerase II activity results in a higher rate of misrepair, probably due to some compromised G2 phase processes involving the WRN protein.     

Role for the Werner syndrome protein in the promotion of tumor cell growth

Werner syndrome (WS) is a premature aging and cancer-prone disease caused by loss of the RecQ helicase WRN protein. Cultured WS fibroblasts display high genomic instability and senesce prematurely. Epigenetic inactivation of the WRN gene occurs in numerous tumor types, in which WRN demonstrates tumor suppressor-like activity (Agrelo et al., 2006). However, the role of WRN in tumors that express WRN protein is unknown. Here we report that the inhibition of WRN expression strongly impairs growth of 12 out of 15 cancer cell lines tested. For those cell lines in which WRN depletion induced high cell death, the majority of the surviving proliferative clones exhibited WRN expression. Growth arrest induced by WRN depletion was characterized by an accumulation of cells in the G2/M cell cycle phases and an increase in DNA damage. Importantly, WRN depletion inhibited tumor growth in vivo in SCID mouse xenograft models. Altogether, these findings support a dual role for WRN in tumorigenesis; tumor suppressor-like activity in tumors with WRN inactivation and the promotion of proliferation and survival in tumors that express WRN. These findings suggest a possible therapeutic role for WRN as an anti-cancer target, and highlight the importance of WRN protein status for tumorigenesis and clinical treatments of patients.     

In vivo function of the conserved non-catalytic domain of Werner syndrome helicase in DNA replication

Werner syndrome is a genetic disorder characterized by genomic instability, elevated recombination and replication defects. The WRN gene encodes a RecQ helicase whose function(s) in cellular DNA metabolism is not well understood. To investigate the role of WRN in replication, we examined its ability to rescue cellular phenotypes of a yeast dna2 mutant defective in a helicase-endonuclease that participates with flap endonuclease 1 (FEN-1) in Okazaki fragment processing. Genetic complementation studies indicate that human WRN rescues dna2-1 mutant phenotypes of growth, cell cycle arrest and sensitivity to the replication inhibitor hydroxyurea or DNA damaging agent methylmethane sulfonate. A conserved non-catalytic C-terminal domain of WRN was sufficient for genetic rescue of dna2-1 mutant phenotypes. WRN and yeast FEN-1 were reciprocally co-immunoprecipitated from extracts of transformed dna2-1 cells. A physical interaction between yeast FEN-1 and WRN is demonstrated by yeast FEN-1 affinity pull-down experiments using transformed dna2-1 cells extracts and by ELISA assays with purified recombinant proteins. Biochemical analyses demonstrate that the C-terminal domain of WRN or BLM stimulates FEN-1 cleavage of its proposed physiological substrates during replication. Collectively, the results suggest that the WRN-FEN-1 interaction is biologically important in DNA metabolism and are consistent with a role of the conserved non-catalytic domain of a human RecQ helicase in DNA replication intermediate processing.     

DNA helicase deficiencies associated with cancer predisposition and premature ageing disorders

Deficiency in a helicase of the RecQ family is found in at least three human genetic disorders associated with cancer predisposition and/or premature ageing. The RecQ helicases encoded by the BLM, WRN and RECQ4 genes are defective in Bloom's, Werner's and Rothmund-Thomson syndromes, respectively. Cells derived from individuals with these disorders in each case show inherent genomic instability. Recent studies have demonstrated direct interactions between these RecQ helicases and human nuclear proteins required for several aspects of chromosome maintenance, including p53, BRCA1, topoisomerase III, replication protein A and DNA polymerase delta. Here, we review this network of protein interactions, and the clues that they present regarding the potential roles of RecQ family members in DNA repair, replication and/or recombination pathways.     

Homologous recombination and maintenance of genome integrity: cancer and aging through the prism of human RecQ helicases

Homologous recombination (HR) is a genetic mechanism in somatic cells that repairs DNA double-strand breaks and restores productive DNA synthesis following disruption of replication forks. Although HR is indispensable for maintaining genome integrity, it must be tightly regulated to avoid harmful outcomes. HR-associated genomic instabilities arise in three human genetic disorders, Bloom syndrome (BS), Werner syndrome (WS), and Rothmund-Thomson syndrome (RTS), which are caused by defects in three individual proteins of the RecQ family of helicases, BLM, WRN, and RECQL4, respectively. Cells derived from persons with these syndromes display varying types of genomic instability as evidenced by the presence of different kinds of chromosomal abnormalities and different sensitivities to DNA damaging agents. Persons with these syndromes exhibit a variety of developmental defects and are predisposed to a wide range of cancers. WS and RTS are further characterized by premature aging. Recent research has shown many connections between all three proteins and the regulation of excess HR. Here, we illustrate the elaborate networks of BLM, WRN, and RECQL4 in regulating HR, and the potential mechanistic linkages to cancer and aging.     

WRN participates in translesion synthesis pathway through interaction with NBS1

Werner syndrome (WS), caused by mutation of the WRN gene, is an autosomal recessive disorder associated with premature aging and predisposition to cancer. WRN belongs to the RecQ DNA helicase family, members of which play a role in maintaining genomic stability. Here, we demonstrate that WRN rapidly forms discrete nuclear foci in an NBS1-dependent manner following DNA damage. NBS1 physically interacts with WRN through its FHA domain, which interaction is important for the phosphorylation of WRN. WRN subsequently forms DNA damage-dependent foci during the S phase, but not in the G1 phase. WS cells exhibit an increase in spontaneous focus formation of polη and Rad18, which are important for translesion synthesis (TLS). WRN also interacts with PCNA in the absence of DNA damage, but DNA damage induces the dissociation of PCNA from WRN, leading to the ubiquitination of PCNA, which is essential for TLS. This dissociation correlates with ATM/NBS1-dependent degradation of WRN. Moreover, WS cells show constitutive ubiquitination of PCNA and interaction between PCNA and Rad18 E3 ligase in the absence of DNA damage. Taken together, these results indicate that WRN participates in the TLS pathway to prevent genomic instability in an ATM/NBS1-dependent manner.     

Werner syndrome and mutations of the WRN and LMNA genes in France

Werner syndrome (WS) is a pleiotropic disease of premature aging involving short stature, tight, atrophied, and/or ulcerated skin; a characteristic 'birdlike' facies and high, squeaky or hoarse voice; premature greying and thinning of the hair; and early onset cataracts. Additional common symptoms include diabetes mellitus, hypogonadism, osteoporosis, osteosclerosis of the digits, soft tissue calcification, premature atherosclerosis, rare or multiple neoplasms, malformed teeth, and flat feet. Diagnosis can be difficult due to the variable presentation and rarity of the disorder. Transmission is usually autosomal recessive. The WS gene, WRN, is member of the RecQ DNA helicase family. Biallelic mutations of WRN are responsible for most patients. Although heterozygous missense mutations in the LMNA gene have been observed in severely affected WS patients, this only accounts for a small fraction of non-WRN patients. Eighteen WS cases were referred to us for molecular analysis. Eleven had definite and three had probable WS according to the University of Washington Registry clinical criteria. All exons of the WRN gene and their splice junctions were sequenced. Of the fourteen definite or probable cases, 11 had one or more WRN mutation. Thirteen different mutations were found, and ten of these were previously undescribed. There were few phenotypic differences between patients with WRN mutation(s) and those who met clinical criteria though lacking WRN mutations. However, patients with mutations tended to have more symptoms overall, and mutations were not observed in the two cases with cardiomyopathy.     

WRN mutations in Werner syndrome

Werner syndrome (WS) is one of a group of human genetic diseases that have recently been linked to deficits in cellular helicase function. We review the spectrum of WS-associated WRN mutations, the organization and potential functions of the WRN protein, and potential mechanistic links between the loss of WRN function and pathogenesis of the WS clinical and cellular phenotypes.     

Loss of Werner syndrome protein function promotes aberrant mitotic recombination

The chromosome 8p11-12 Werner syndrome (WRN ) locus encodes a RecQ helicase protein of unknown function that possesses both 3' --> 5' helicase and 3' --> 5' exonuclease activities. We show that WRN cell lines display a marked reduction in cell proliferation following mitotic recombination, and generate few viable gene conversion-type recombinants. These findings indicate that WRN plays a role in mitotic recombination, and that a loss of WRN function may promote genetic instability and disease via recombination-initiated mitotic arrest, cell death, or gene rearrangement.     

The N-terminal domain of the large subunit of human replication protein A binds to Werner syndrome protein and stimulates helicase activity

Werner syndrome (WS) is a recessive inherited human disease characterized by the early onset of aging. The gene mutated in WS encodes a DNA helicase that unwinds the double helical structure of DNA in the 3'-->5' direction as well as a 3'-->5' exonuclease. Our previous studies indicated that the activity of Werner syndrome helicase (WRN) could be stimulated by human replication protein A (hRPA), a heterotrimeric single-stranded DNA binding protein. We now localize the interaction between WRN and hRPA by measuring the stimulation of helicase activity and the binding of WRN by hRPA and its derivatives. The large subunit of hRPA (hRPA70) stimulates WRN helicase to the same extent as the hRPA heterotrimer, whereas the dimer of the two smaller subunits (hRPA 32.14) does not stimulate. By examining hRPA70 mutants with progressive deletions from either the C- or N-terminus, we found that the domain responsible for stimulation lies in the N-terminal half of the protein. By using enzyme-linked immunosorbent assay (ELISA) to examine physical interaction between WRN and the same deletion mutants, we found that the WRN-binding motif is located within amino acids 100-300 and overlaps with the single-stranded DNA binding domain (amino acids 150-450). We suggest that hRPA, by engaging in both protein-protein and protein-DNA interactions, facilitates unwinding events catalyzed by WRN helicase during DNA synthetic processes. These data should help further elucidation of the molecular mechanisms of genetic instability and premature aging phenotypes manifested by WS.     

Cellular dynamics and modulation of WRN protein is DNA damage specific

The human premature aging protein Werner (WRN), deficient in Werner syndrome (WS), is localized mainly to the nucleolus in many cell types. DNA damage or replication arrest causes WRN to redistribute from the nucleolus to the nucleoplasm into discrete foci. In this study, we have investigated DNA damage specific cellular redistribution of WRN. In response to agents causing DNA double strand breaks or DNA base damage, WRN is re-distributed from the nucleolus to the nucleoplasm in a reversible manner. However, after ultraviolet (UV) irradiation such redistribution of WRN is largely absent. We also show that WRN is associated with the insoluble protein fraction of cells after exposure to various kinds of DNA damage but not after UV irradiation. Further, we have studied the DNA damage specific post-translational modulation of WRN. Our results show that WRN is acetylated after mytomycin C or methyl methane-sulfonate treatment, but not after UV irradiation. Also, DNA damage specific phosphorylation of WRN is absent in UV irradiated cells. Inhibition of phosphorylation fails to restore WRN localization. Thus, our results suggest that the dynamics of WRN protein trafficking is DNA damage specific and is related to its post-translational modulation. The results also indicate a preferred role of WRN in recombination and base excision repair rather than nucleotide excision repair.     

p53-Mediated apoptosis is attenuated in Werner syndrome cells

The WRN DNA helicase is a member of the DExH-containing DNA helicase superfamily that includes XPB, XPD, and BLM. Mutations in WRN are found in patients with the premature aging and cancer susceptibility syndrome known as Werner syndrome (WS). p53 binds to the WRN protein in vivo and in vitro through its carboxyl terminus. WS fibroblasts have an attenuated p53- mediated apoptotic response, and this deficiency can be rescued by expression of wild-type WRN. These data support the hypothesis that p53 can induce apoptosis through the modulation of specific DExH-containing DNA helicases and may have implications for the cancer predisposition observed in WS patients.     

The Werner syndrome protein at the crossroads of DNA repair and apoptosis

Werner syndrome (WS) is a premature aging disease characterized by genetic instability. WS is caused by mutations in a gene encoding for a 160 kDa nuclear protein, the Werner syndrome protein (WRN), which has exonuclease and helicase activities. The mechanism whereby WRN controls genome stability and life span is not known. Over the last few years, WRN has become the focus of intense investigation by a growing number of scientists. The studies carried out by many laboratories have provided a wealth of new information about the functional properties of WRN and its cellular partners. This review focuses on recent findings that demonstrate a functional interaction between WRN and two factors that bind to DNA breaks, Ku and poly(ADP-ribose) polymerase 1, and discuss how these interactions can influence fundamental cellular processes such as DNA repair, apoptosis and possibly regulate cell senescence and organismal aging.