Abstract
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.
Highlights
Werner Syndrome (WS),1 characterized by premature aging and genomic instability [1], is a result of mutations in the WS gene
The bimolecular tetraplex complex formed between two molecules of primed32P-template, schematically illustrated in Fig. 1B and designated as GЈ2 primer-template, was extracted from the gel and served as a substrate for WRN helicase as well as a template for DNA synthesis by DNA polymerases
The finding that stimulation of pol ␦ activity by WRN occurs in the absence of the pol ␦ accessory factor, proliferating cell nuclear antigen [28], suggested that WRN may not participate in processive DNA replication
Summary
Materials and Enzymes [␥-32P]ATP (ϳ3000 Ci/mmol) was purchased from PerkinElmer Life Sciences. Ultrapure deoxyribonucleoside triphosphates (dNTPs) were purchased from Promega Corp. Recombinant hexa-His-tagged WRN protein was purified to Ͼ90% homogeneity by the protocol published by Shen et al [16]. Approximate concentrations of WRN protein were determined from Coomassiestained SDS-polyacrylamide gels using bovine serum albumin as a standard. S. cerevisiae DNA pol ␦ and pol ␦* were purified to homogeneity as described [44]; concentrations of pol ␦ and pol ␦* were determined spectrophotometrically at A280. Human DNA polymerase ␣-primase complex (pol ␣) and human DNA polymerase ⑀ (pol ⑀) were the generous gifts of Dr Teresa Wang (Stanford University, Stanford, CA) and Dr Stuart Linn (University of California, Berkeley, CA), respectively
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