The human premature aging disorder Werner syndrome (WS) is associated with a large number of symptoms displayed in normal aging. The WRN gene product, a DNA helicase, has been previously shown to unwind short DNA duplexes (<53 base pairs) in a reaction stimulated by single-stranded DNA-binding proteins. We have studied the helicase activity of purified WRN protein on a variety of DNA duplex substrates to characterize the unwinding properties of the enzyme in greater detail. WRN helicase can catalyze unwinding of long duplex DNA substrates up to 849 base pairs in a reaction dependent on human replication protein A (hRPA). Escherichia coli SSB and bacteriophage T4 gene 32 protein (gp32) completely failed to stimulate WRN helicase to unwind long DNA duplexes indicating a specific functional interaction between WRN and hRPA. So far, there have been no reports of any physical interactions between WRN helicase and other proteins. In support of the functional interaction, we demonstrate a direct interaction between WRN and hRPA by coimmunoprecipitation of purified proteins. The physical and functional interaction between WRN and hRPA suggests that the two proteins may function together in vivo in a pathway of DNA metabolism such as replication, recombination, or repair.

The uvrA, uvrB, and uvrC genes control excision repair in Escherichia coli. Cells with mutations in any of these three genes cannot repair DNA by nucleotide excision. When the purified gene products-the UvrA, UvrB, and UvrC proteins-are mixed together, an excision nuclease is formed that incises on both sides of the damaged nucleotide in an ATP-dependent reaction; it has been presumed that the excision nuclease was an ABC complex containing all three Uvr proteins. To determine the stoichiometry of the subunits in the enzyme, we conducted hydrodynamic studies with mixtures of the subunits with or without DNA substrate. We found that without DNA the UvrA subunit is a dimer and that when UvrB protein is also present, a (UvrA)2(UvrB), complex forms.Without DNA no detectable interaction of either the UvrA or UvrB subunits or the (UvrA)2(UvrB), complex with the UvrC subunit occurs. Unexpectedly, with UV-irradiated DNA, the UvrA/UvrB ratio in isolated DNA-protein complexes is variable, and the ratio becomes infinitesimally low as the UvrA concentration in the reaction mixture decreases. Under conditions of saturating UvrB protein approximately one UvrB molecule binds to DNA per damaged site in a reaction that requires catalytic amounts of UvrA subunit. Addition of UvrC protein to purified UvrB-DNA complexes results in rapid incision of the DNA, presumably catalyzed by an excision nuclease containing only UvrB and UvrC subunits.The UvrA, UvrB, and UvrC proteins, referred to collectively as "ABC excinuclease," initiate nucleotide excision repair in Escherichia coli by incising seven bases 5' and four bases 3' to nucleotide mono-and diadducts in DNA (1) We have conducted physical studies to determine the stoichiometry of the subunits in preincision complexes of the Uvr proteins in the absence and presence of DNA. We found that UvrA dimerizes and, in an ATP-dependent reaction, associates with UvrB to form a (UvrA)2(UvrB), complex in the absence of DNA. More importantly, we have determined that UvrA, possibly as the (UvrA)2(UvrB), complex, delivers UvrB onto damaged DNA and then dissociates from the UvrB-DNA complex. Upon addition of UvrC, the DNA in the UvrB-DNA complex is incised in a manner typical of the so-called ABC excinuclease. Thus, our data indicate that UvrA is essential for the formation of the nucleotide excision nuclease complex, but it is not involved in the incision reactions. MATERIALS AND METHODSEnzymes and Substrates. The UvrA, UvrB, and UvrC proteins were purified to >909% homogeneity as described (4). Phage T4 endonuclease V was purchased from Applied Genetics (Freeport, NY). Protein standards for hydrodynamic analyses were from Bio-Rad and M13mp19RF DNA was from Boehringer Mannheim. 3H-labeled pBR322 DNA (1.1 x 104 cpm/,ug) was purified from E. coli AB2487/ pBR322 by Sarkosyl lysis followed by two cesium chloride/ ethidium bromide density centrifugations. When indicated, DNA was damaged by UV irradiation (25-375 J/m2) by using a Sylvania germicidal lamp (254 nm). The number of pyrimidine dimers...

RecQ helicases are critical for maintaining genomic integrity. In this study, we show that three RecQ members (WRN, deficient in the Werner syndrome; BLM, deficient in the Bloom syndrome; and Drosophila melanogaster RecQ5b (dmRecQ5b)) possess a novel strand pairing activity. Furthermore, each of these enzymes combines this strand pairing activity with its inherent DNA unwinding capability to perform coordinated strand exchange. In this regard, WRN and BLM are considerably more efficient than dmRecQ5b, apparently because dmRecQ5b lacks conserved sequences C-terminal to the helicase domain that contribute to DNA binding, strand pairing, and strand exchange. Based on our findings, we postulate that certain RecQ helicases are structurally designed to accomplish strand exchange on complex replication and recombination intermediates. This is highly consistent with proposed roles for RecQ members in DNA metabolism and the illegitimate recombination and cancer-prone phenotypes associated with RecQ defects.RecQ family members are 3Ј to 5Ј DNA helicases that function in genome maintenance. Prokaryotes and lower eukaryotes have one RecQ helicase, whereas humans have five (RECQ1, BLM, WRN, RECQ4, and RECQ5). Notably, Bloom, Werner, and Rothmund-Thomson syndromes are autosomal recessive diseases caused by loss of function of BLM, WRN, and RECQ4, respectively (1-3). Cancer incidence is markedly elevated in these syndromes (particularly in Bloom syndrome), although each has a distinct clinical phenotype. Werner syndrome is especially notable for the accelerated development of certain aging characteristics, including graying and loss of hair, atherosclerosis, cataracts, diabetes, and osteoporosis (4, 5). Thus, these diseases serve as excellent model systems to investigate the role of genetic changes in cancer and specific age-related problems. At the cellular level, deficiencies in RecQ family members result in increased chromosomal abnormalities due to illegitimate recombination, suggesting functions for these helicases in recombination repair or resolution of replication blockage (6 -11). Accordingly, RecQ helicases preferentially bind to and unwind substrates mimicking replication and recombination intermediates (12-18). Recent biochemical and structural studies (19 -22) also indicate that specific RecQ helicases, including WRN and BLM, contain multiple DNA-binding domains. Hypothetically, it would be advantageous for proteins that act on the complex three-and four-stranded DNA structures typically associated with replication and recombination to have multiple DNA-binding domains.The predicted roles of RecQ helicases combined with their multiple DNA-binding domains suggested that these enzymes might have, in addition to their requisite unwinding capability, other recombination functions such as strand pairing and strand exchange. The experiments described below indicate that three RecQ members (WRN, BLM, and Drosophila melanogaster RecQ5b (dmRecQ5b) 1 ) individually possess a novel strand pairing activity that functions in s...

In addition to increased DNA-strand exchange, a cytogenetic feature of cells lacking the RecQ-like BLM helicase is a tendency for telomeres to associate. We also report additional cellular and biochemical evidence for the role of BLM in telomere maintenance. BLM co-localizes and complexes with the telomere repeat protein TRF2 in cells that employ the recombination-mediated mechanism of telomere lengthening known as ALT (alternative lengthening of telomeres). BLM co-localizes with TRF2 in foci actively synthesizing DNA during late S and G2/M; co-localization increases in late S and G2/M when ALT is thought to occur. Additionally, TRF1 and TRF2 interact directly with BLM and regulate BLM unwinding activity in vitro. Whereas TRF2 stimulates BLM unwinding of telomeric and non-telomeric substrates, TRF1 inhibits BLM unwinding of telomeric substrates only. Finally, TRF2 stimulates BLM unwinding with equimolar concentrations of TRF1, but not when TRF1 is added in molar excess. These data suggest a function for BLM in recombination-mediated telomere lengthening and support a model for the coordinated regulation of BLM activity at telomeres by TRF1 and TRF2.

The premature aging and cancer-prone diseases Werner and Bloom syndromes are caused by loss of function of WRN and BLM proteins, respectively. At the cellular level, WRN or BLM deficiency causes replication abnormalities, DNA damage hypersensitivity, and genome instability, suggesting that these proteins might participate in resolution of replication blockage. Although WRN and BLM are helicases belonging to the RecQ family, both have been recently shown to also facilitate pairing of complementary DNA strands. In this study, we demonstrate that both WRN and BLM (but not other selected helicases) can coordinate their unwinding and pairing activities to regress a model replication fork substrate. Notably, fork regression is widely believed to be the initial step in responding to replication blockage. Our findings suggest that WRN and/or BLM might regress replication forks in vivo as part of a genome maintenance pathway, consistent with the phenotypes of WRN- and BLM-deficient cells.

Werner syndrome is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutations of the WRN gene. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Sir2, an NAD-dependent histone deacetylase, has been proven to extend life span in yeast and Caenorhabditis elegans. Mammalian Sir2 (SIRT1) has also been found to regulate premature cellular senescence induced by the tumor suppressors PML and p53. SIRT1 plays an important role in cell survival promoted by calorie restriction. Here we show that SIRT1 interacts with WRN both in vitro and in vivo; this interaction is enhanced after DNA damage. WRN can be acetylated by acetyltransferase CBP/p300, and SIRT1 can deacetylate WRN both in vitro and in vivo. WRN acetylation decreases its helicase and exonuclease activities, and SIRT1 can reverse this effect. WRN acetylation alters its nuclear distribution. Down-regulation of SIRT1 reduces WRN translocation from nucleoplasm to nucleoli after DNA damage. These results suggest that SIRT1 regulates WRN-mediated cellular responses to DNA damage through deacetylation of WRN. Werner syndrome (WS)3 is a human autosomal recessive disorder that displays symptoms of premature aging, including graying and loss of hair, wrinkling and ulceration of skin, atherosclerosis, osteoporosis, and cataracts. WS patients also exhibit an increased incidence of diabetes mellitus type 2, hypertension, and are highly disposed to cancers (1). WS results from mutation of the WRN gene, a member of the RecQ DNA helicase family (2). Mutations in other family members, BLM and RECQ4, are responsible for the two other cancer-prone and premature aging syndromes, Bloom (3) and Rothmund-Thomson (4), respectively. Consistent with other RecQ helicases, WRN protein possesses 3Ј to 5Ј DNA helicase activity; however, it is the only human RecQ member to also have a 3Ј to 5Ј exonuclease activity. Although its physiological substrate is not yet clear, WRN appears to preferentially act on replication and recombination structures. Cells from WS patients show premature replicative senescence compared with cells derived from normal individuals (5). WS cells also show hypersensitivity to selected DNA-damaging agents including 4-nitroquinoline-1-oxide (4NQO; 6), topoisomerase inhibitors (7), and certain DNA cross-linking agents (8). Compared with normal cells, WS cells also exhibit increased genomic instability including higher levels of DNA deletions, translocations, and chromosomal breaks (9, 10), suggesting that WRN plays an important role in one or more genome maintenance pathways (11).WRN protein shows dynamic relocalization within the nucleus under different conditions of growth. The WRN protein localizes to the nucleoli in a variety of cell types (12), and this localization is modulated by DNA damage and cell cycle. Upon serum starvation or treatment with hydroxyurea (HU), aphidicolin, 4NQO, etoposide, or camptothecin, WRN migrates from nucleoli to discrete nuclear foci (13-17). ...

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.

Mutations in the WRN gene result in Werner syndrome, an autosomal recessive disease in which many characteristics of aging are accelerated. A probable role in some aspect of DNA metabolism is suggested by the primary sequence of the WRN gene product. A recombinant His-tagged WRN protein (WRNp) was overproduced in insect cells using the baculovirus system and purified to near homogeneity by several chromatographic steps. This purification scheme removes both nuclease and topoisomerase contaminants that persist following a single Ni(2+)affinity chromatography step and allows for unambiguous interpretation of WRNp enzymatic activities on DNA substrates. Purified WRNp has DNA-dependent ATPase and helicase activities consistent with its homology to the RecQ subfamily of proteins. The protein also binds with higher affinity to single-stranded DNA than to double-stranded DNA. However, WRNp has no higher affinity for various types of DNA damage, including adducts formed during 4NQO treatment, than for undamaged DNA. Our results confirm that WRNp has a role in DNA metabolism, although this role does not appear to be the specific recognition of damage in DNA.