Published online 23 August 2007 Nucleic Acids Research, 2007, Vol. 35, No. 17 5729–5747 doi:10.1093/nar/gkm561 Replication fork regression in vitro by the Werner syndrome protein (WRN): Holliday junction formation, the effect of leading arm structure and a potential role for WRN exonuclease activity Amrita Machwe 1 , Liren Xiao 1 , Robert G. Lloyd 2 , Edward Bolt3 and David K. Orren1,* 1Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536, 2Institute of Genetics and 3The School of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham NG 72UH, UK Received May 4, 2007; Revised June 7, 2007; Accepted July 7, 2007 ABSTRACT The premature aging and cancer-prone disease Werner syndrome stems from loss of WRN protein function. WRN deficiency causes replication abnormalities, sensitivity to certain genotoxic agents, genomic instability and early replicative senescence in primary fibroblasts. As a RecQ heli- case family member, WRN is a DNA-dependent ATPase and unwinding enzyme, but also possesses strand annealing and exonuclease activities. RecQ helicases are postulated to participate in pathways responding to replication blockage, pathways possibly initiated by fork regression. In this study, a series of model replication fork substrates were used to examine the fork regres- sion capability of WRN. Our results demonstrate that WRN catalyzes fork regression and Holliday junction formation. This process is an ATP- dependent reaction that is particularly efficient on forks containing single-stranded gaps of at least 11–13 nt on the leading arm at the fork junction. Importantly, WRN exonuclease activity, by digesting the leading daughter strand, enhances regression of forks with smaller gaps on the leading arm, thus creating an optimal structure for regression. Our results suggest that the multiple activities of WRN cooperate to promote replication fork regression. These findings, along with the estab- lished cellular consequences of WRN deficiency, strongly support a role for WRN in regression of blocked replication forks. INTRODUCTION Werner syndrome (WS) is a rare, autosomal recessive disease characterized by early onset and increased frequency of many phenotypes normally associated with human aging including graying and loss of hair, wrinkling and ulceration of skin, cancer, atherosclerosis, cataracts, osteoporosis, diabetes and hypertension (1,2). Intriguingly, all of these phenotypes result from loss of function of a single gene product, WRN, belonging to the RecQ family of DNA helicases (3) that includes the prototype RecQ in E. coli, Sgs1 in S. cerevisiae, Rqh1 in S. pombe and four other family members in humans. Importantly, the highly cancer-prone Bloom syndrome (BS) is caused by mutations in human RecQ family member BLM (4), while Rothmund–Thomson (RTS), RAPADILINO and Baller–Gerold syndromes are caused by diﬀerent mutations in family member RECQL4 (5–7). The RECQL4-related syndromes are collectively charac- terized by abnormalities in skeletal development and skin pigmentation (poikiloderma), but RTS also shows an elevated incidence of osteosarcoma. At the cellular level, loss of function of a RecQ family member generally results in increased spontaneous and damage-induced chromoso- mal aberrations, suggesting crucial functions for these proteins in maintaining large-scale genome stability. In agreement with this notion, WRN-deﬁcient cells have higher frequencies of chromosomal deletions, insertions and translocations and are more sensitive to selected DNA damaging agents (including replication inhibitors, topo- isomerase I inhibitors and interstrand crosslinking agents) than cells derived from normal individuals (8–13). Moreover, primary ﬁbroblasts from individuals with WS rapidly undergo senescence in culture, apparently *To whom correspondence should be addressed. Tel: +859 323 3612; Fax: +859 323 1059; Email: dkorre2@uky.edu 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.