Werner syndrome (WS) is an uncommon autosomal recessive disorder characterized by premature aging. The clinical manifestations of WS, including atherosclerosis and osteoporosis, appear early in adulthood, and death in the fourth to sixth decade commonly ensues from myocardial infarction or cancer. In accord with the aging phenotype, cells from WS patients have a reduced replicative life span in culture. Genomic instability is observed at the cytogenetic level in the form of chromosome breaks and translocations and at the molecular level by multiple large deletions. The Werner syndrome gene (WRN) has recently been cloned. The predicted product is a 1,432-amino-acid protein whose central domain is homologous to members of the RecQ family of DNA helicases. Such homology does not necessarily mean that WRN encodes an active helicase. For example, the Saccharomyces cerevisiae RAD26 gene protein and the human transcription-repair coupling factor CSB (Cockayne syndrome 8) are highly homologous to known helicases, yet neither encodes an active helicase. Moreover, the Bloom's syndrome gene (BLM), discovered before WRN, is also homologous to the RecQ family of DNA helicases, though we still await demonstration that it encodes an active helicase. Here we report that the WS protein does indeed catalyze DNA unwinding.

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 3 5 DNA helicase and 3 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 G2 bimolecular tetraplex structures of the fragile X expanded sequence, d(CGG) n , effectively impede synthesis by three eukaryotic replicative DNA polymerases (pol): pol ␣, pol ␦, and pol ⑀. The constraints imposed on pol ␦-catalyzed synthesis are relieved, however, by WRN; WRN facilitates pol ␦ to traverse these template secondary structures to synthesize full-length DNA products. The alleviatory effect of WRN is limited to pol ␦; neither pol ␣ nor pol ⑀ can traverse template d(CGG) n hairpin and tetraplex structures in the presence of WRN. Alleviation of pausing by pol ␦ is observed with Escherichia coli RecQ but not with UvrD helicase, suggesting a concerted action of RecQ helicases and pol ␦. Our findings suggest a possible role of WRN in rescuing pol ␦-mediated replication at forks stalled by unusual DNA secondary structures.Werner Syndrome (WS), 1 characterized by premature aging and genomic instability (1), is a result of mutations in the WS gene. The polypeptide encoded by the WS gene, WRN, contains a central seven-motif domain shared by DNA helicases of the RecQ family (2). This family of DNA helicases is represented by Escherichia coli RecQ (3), Saccharomyces cerevisiae Sgs-1 (4), Schizosaccharomyces pombe Rqh1 (5), Xenopus laevis FFA-1 (6), and human RecQL (7), BLM (8), and RecQ4 and RecQ5 proteins (9). Multiple RecQ DNA helicases have also been identified in Drosophila melanogaster (10) and Arabidopsis thaliana (11). WRN is distinct from other members of the RecQ helicase family in that it also includes an N-terminal exonuclease domain (12)(13)(14). Indeed, recombinant WRN protein has been shown to possess, in addition to an ATP-dependent 3Ј 3 5Ј DNA helicase activity, an intrinsic 3Ј 3 5Ј DNA exonuclease activity (15, 16).WRN helicase exhibits several characteristic features. 1) Unwinding of double-stranded DNA requires a 3Ј singlestranded DNA tail, which presumably serves as a helicase loading DNA stretch (17, 18). 2) WRN exhibits low processivity such that the enzyme is capable of unwinding only short duplex regions Ͻ25 nt in length.3) The processivity of WRN can be increased by the single-stranded DNA-binding protein, human replication protein A (19); in its presence, WRN unwinds duplex DNA tracts as long as 800 nt (20). 4) WRN can unwind alternate DNA structures, including DNA tetraplexes (21), four-way Holliday junctions (22), and triplex DNA (23).A large body of evidence implicates WRN and its family members in replication. The prolonged S-phase of WS cells (24,25), their sensitivity to the S-phase-specific topoisomerase I inhibitor cam...

In addition to its DNA helicase activity, Werner syndrome protein (WRN) also possesses an exonuclease activity (Shen, J.-C. , Gray, M. D., Kamath-Loeb, A. S., Fry, M., Oshima, J., and Loeb, L. A. (1998) J. Biol. Chem. 273, 34139 -34144). Here we describe the properties of nearly homogeneous WRN exonuclease. WRN exonuclease hydrolyzes a recessed strand in a partial DNA duplex but does not significantly digest single-stranded DNA, blunt-ended duplex, or a protruding strand of a partial duplex. Although DNA is hydrolyzed in the absence of nucleoside triphosphates, nuclease activity is markedly stimulated by ATP, dATP, or CTP. WRN exonuclease digests DNA with a 3 3 5 directionality to generate 5-dNMP products, and DNA strands terminating with either a 3-OH or 3-PO 4 group are hydrolyzed to similar extents. A recessed DNA strand with a single 3-terminal mismatch is hydrolyzed more efficiently by WRN than one with a complementary nucleotide, but the enzyme fails to hydrolyze a DNA strand terminating with two mismatched bases. WRN exonuclease is distinguished from known mammalian DNA nucleases by its covalent association with a DNA helicase, preference for a recessed DNA strand, stimulation by ATP, ability to equally digest DNA with 3-OH or 3-PO 4 termini, and its preferential digestion of DNA with a single 3-terminal mismatch.Werner Syndrome (WS) 1 is a recessive inherited disease characterized by genetic instability and aging in early adulthood (1, 2). The gene defective in WS, WRN, encodes a 3Ј 3 5Ј RecQ-like DNA helicase that unwinds DNA in an ATP-dependent manner (3-5). Mutations in WRN are invariably found in patients exhibiting the clinical symptoms of WS (6, 7). These include atherosclerosis, osteoporosis, diabetes mellitus, and bilateral cataracts, as well as an unusually high incidence of tumors of non-epithelial cell origin. At the cellular level, WS cells are characterized by chromosomal translocations, large DNA deletions, elevated rates of homologous recombination, defective maintenance of telomeres, and a prolonged S-phase of DNA synthesis (8 -15).In the preceding paper (16), we reported the identification of a novel exonuclease activity in WRN. We used molecular genetic, biochemical, and immunochemical methods to establish that the exonuclease, like the DNA helicase, is integral to WRN. Although the two activities are expressed in the same polypeptide in the wild-type protein, they can be uncoupled from each other by introducing mutations separately in each of the two domains. In patients, mutations in WRN are not necessarily located in the helicase domain. They are found throughout the gene and invariably introduce stop codons or deletions (6, 7). It has been argued that many mutations obliterate the nuclear localization signal and that lack of localization may be important in the pathogenesis of WS (17, 18). This lack of nuclear localization would result in deficits of both helicase and exonuclease activities.To gain a better understanding of the functions of the WRN exonuclease, we have studied its...

Werner Syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability. The WS gene encodes a 1,432-amino acid polypeptide (WRN) with a central domain homologous to the RecQ family of DNA helicases. Purified WRN unwinds DNA with 335 polarity, and also possesses 335 exonuclease activity. Elucidation of the physiologic function(s) of WRN may be aided by the identification of WRN-interacting proteins. We show here that WRN functionally interacts with DNA polymerase ␦ (pol ␦), a eukaryotic polymerase required for DNA replication and DNA repair. WRN increases the rate of nucleotide incorporation by pol ␦ in the absence of proliferating cell nuclear antigen (PCNA) but does not stimulate the activity of eukaryotic DNA polymerases ␣ or , or a variety of other DNA polymerases. Moreover, we show that functional interaction with WRN is mediated through the third subunit of pol ␦: i.e., Pol32p of Saccharomyces cerevisae, corresponding to the recently identified p66 subunit of human pol ␦. Absence of the third subunit abrogates stimulation by WRN, and stimulation is restored by reconstituting the three-subunit enzyme. Our findings suggest that WRN may facilitate pol ␦-mediated DNA replication and͞or DNA repair and that disruption of WRN-pol ␦ interaction in WS cells may contribute to the previously observed S-phase defects and͞or the unusual sensitivity to a limited number of DNA damaging agents. Werner Syndrome (WS) is an autosomal, recessive progeroid disorder characterized by genomic instability (1). Patients with WS prematurely exhibit age-related conditions such as atherosclerosis, osteoporosis, diabetes mellitus, and bilateral cataracts. Additionally, they show an increased incidence of cancers of non-epithelial cell lineage (2). Genetic instability in WS is manifested at the chromosomal level by breaks and rearrangements, and at the DNA level by multiple, large deletions (3-5).The WS gene encodes a 1,432-amino acid polypeptide (WRN) containing a domain homologous to the RecQ family of DNA helicases (6). This family is represented by the prototypical Escherichia coli RecQ (7), Saccharomyces cerevisiae Sgs1 (8), Schizosaccharomyces pombe Rqh1 (9), Xenopus laevis FFA-1 (Focus forming activity) (10), and the human proteins RecQL (11), BLM (the product of the Bloom's syndrome gene) (12), RecQ4 (the product of the Rothmund-Thomson syndrome gene) and RecQ5 (13,14).Several RecQ family members, including WRN, have been purified and shown to possess 3Ј35Ј DNA unwinding activity in vitro (15)(16)(17)(18)(19)(20). WRN helicase activity exhibits several features: First, unwinding of duplex DNA is ATP-dependent and typically requires a 3Ј single-stranded tail (19,21). Second, the activity is nonprocessive, limited to unwinding small stretches (Ͻ25 nt) of duplex DNA. Third, processivity is increased by ssDNA binding proteins, particularly by human replication protein A (22). In the presence of human replication protein A, WRN can unwind duplex DNA segments as long ...