Homologous Recombination Resolution Defect in Werner Syndrome

Abstract : Werner syndrome (WRN) is an uncommon autosomal recessive disease whose phenotype includes features of premature aging, genetic instability, and an elevated risk of cancer. We used three different experimental strategies to show that WRN cellular phenotypes of limited cell division potential, DNA damage hypersensi-tivity, and defective homologous recombination (HR) are interrelated. WRN cell survival and the generation of viable mitotic recombinant progeny could be rescued by expressing wild-type WRN protein or by expressing the bacterial resolvase protein RusA. The dependence of WRN cellular phenotypes on RAD51-dependent HR pathways was demonstrated by using a dominant-negative RAD51 protein to suppress mitotic recombination in WRN and control cells: the suppression of RAD51-dependent recombination led to significantly improved survival of WRN cells following DNA damage. These results define a physiological role for the WRN RecQ helicase protein in RAD51-dependent HR and identify a mechanistic link between defective recombination resolution and limited cell division potential, DNA damage hypersensitivity, and genetic instability in human somatic cells. Werner syndrome (WRN) was originally identified among adult siblings in a single family, all of whom displayed cataract formation, premature greying and loss of hair, and scleroder-ma-like skin changes (48). Further characterization of the clinical , pathological, and genetic aspects of this syndrome following Otto Werner's initial description has led to the recognition of Werner syndrome as an uncommon autosomal recessive disease whose phenotype includes features of premature aging, genetic instability, and an elevated risk of cancer (13, 18, 39). The WRN gene (also referred to as RECQ3 or RECQL2) was identified by positional cloning in 1996 (51) and was found to encode a 162-kDa member of the human RecQ helicase family with 335 helicase and 335 exonuclease activities. Werner patient mutations truncate the WRN open reading frame and promote loss of the altered protein and both of its associated biochemical activities (4, 28, 42). Mutations in other human RecQ helicase genes have also been identified in patients with two other genetic instability and tumor predisposition syndromes, Bloom syndrome (12) and Rothmund-Thom-son syndrome (24, 26). Recently, a homologous recombination (HR) defect in WRN cell lines was identified that included a 25-fold reduction in the rate of generation of viable recombinant daughter cells together with a shift in molecular recombination products from conversion-type to crossover or "popout"-type recombinants that are normally less frequent (35). These analyses focused on spontaneous mitotic recombintion and did not further define the WRN recombination defect or indicate how HR, cell survival , and the response to DNA damage were interrelated in WRN cells. In the work reported here, three different experimental approaches were used to define the WRN recombina-tion defect and the interrelationship of HR and cell survival following DNA damage in WRN cells. Expression of wild-type WRN protein or the bacterial resolvase protein RusA were both shown to rescue the WRN recombination defect and to improve cell survival following DNA damage. The dependence of WRN cellular phenotypes on RAD51-dependent HR function was demonstrated with a dominant-negative mammalian RAD51 protein (SMRAD51) (22). Expression of SMRAD51 suppressed HR in control and WRN cells as predicted while leading to markedly improved WRN cell survival after DNA damage. These results confirm the presence of an HR defect in WRN cells, more clearly identify the HR stage and likely molecular intermediates or products involved, and demonstrate the interdependence of defective HR, reduced cell division potential , and DNA damage hypersensitivity following a loss of WRN function. These results define a physiological role for WRN. The results also suggest a model for WRN function that explains how WRN loss leads to reduced cell division, DNA damage hypersensitivity, and genetic instability and thus may act to promote disease pathogenesis. MATERIALS AND METHODS Plasmid DNAs. Plasmid DNAs encoding wild-type or K577 M missense mutant forms of human WRN protein have been previously described (28). Oligo-nucleotide-mediated, site-directed mutagenesis (Transformer; Clontech) was used to introduce an E84A substitution in a wild-type WRN open reading frame. An EcoRI-BsrGI fragment containing the E84A substitution was then subcloned into a K577 M WRN expression vector to give a WRN open reading frame and protein that lacked helicase and exonuclease activities. The resulting plasmids were sequence verified and tested for expression by Western blot analysis after transient transfection (see below). The plasmids pPURO and pSMRad51 were kindly provided by Bernard Lopez (CNRS-CEA, Fontenay aux Roses, France) (22). Plasmids pMW400 and pMW436, expressing wild-type and D70N forms of
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Y. Saintigny, K. Makienko, C. Swanson, M. Emond, R. Monnat Jr.. Homologous Recombination Resolution Defect in Werner Syndrome. Molecular and Cellular Biology, American Society for Microbiology, 2002, 22 (20), pp.6971 - 6978. ⟨10.1128/mcb.22.20.6971-6978.2002⟩. ⟨cea-01938070⟩

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