Biochemical characterization of the DNA substrate specificity of Werner syndrome helicase.
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Werner syndrome is a hereditary premature aging disorder characterized by genome instability. The product of the gene defective in WS, WRN, is a helicase/exonuclease that presumably functions in DNA metabolism. To understand the DNA structures WRN acts upon in vivo, we examined its substrate preferences for unwinding. WRN unwound a 3'-single-stranded (ss)DNA-tailed duplex substrate with streptavidin bound to the end of the 3'-ssDNA tail, suggesting that WRN does not require a free DNA end to unwind the duplex; however, WRN was completely blocked by streptavidin bound to the 3'-ssDNA tail 6 nucleotides upstream of the single-stranded/double-stranded DNA junction. WRN efficiently unwound the forked duplex with streptavidin bound just upstream of the junction, suggesting that WRN recognizes elements of the fork structure to initiate unwinding. WRN unwound two important intermediates of replication/repair, a 5'-ssDNA flap substrate and a synthetic replication fork. WRN was able to translocate on the lagging strand of the synthetic replication fork to unwind duplex ahead of the fork. For the 5'-flap structure, WRN specifically displaced the 5'-flap oligonucleotide, suggesting a role of WRN in Okazaki fragment processing. The ability of WRN to target DNA replication/repair intermediates may be relevant to its role in genome stability maintenance. (+info)
WRN helicase accelerates the transcription of ribosomal RNA as a component of an RNA polymerase I-associated complex.
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Werner syndrome (WS) is a rare autosomal recessive genetic disorder causing premature aging. The gene (WRN) responsible for WS encodes a protein homologous to the RecQ-type helicase. WRN has a nucleolar localization signal and shows intranuclear trafficking between the nucleolus and the nucleoplasm. WRN is recruited into the nucleolus when rRNA transcription is reactivated in quiescent cells. Inhibition of mRNA transcription with alpha-amanitin has no effect on nucleolar localization of WRN whereas inhibition of rRNA transcription with actinomycin D releases WRN from nucleoli, suggesting that nucleolar WRN is closely related to rRNA transcription by RNA polymerase I (RPI). A possible function of WRN on rRNA transcription through interaction with RPI is supported by the results described here showing that WRN is co-immunoprecipitated with an RPI subunit, RPA40. Here we show that WS fibroblasts are characterized by a decreased level of rRNA transcription compared with wild-type cells, and that the decreased level of rRNA transcription in WS fibroblasts recovers when wild-type WRN is exogenously expressed. By contrast, exogenously expressed mutant-type WRN lacking an ability to migrate into the nucleolus fails to stimulate rRNA transcription. These results suggest that WRN promotes rRNA transcription as a component of an RPI-associated complex in the nucleolus. (+info)
Werner syndrome diploid fibroblasts are sensitive to 4-nitroquinoline-N-oxide and 8-methoxypsoralen: implications for the disease phenotype.
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The clinical phenotype of Werner Syndrome (WRN) includes features reminiscent of accelerated aging and an increased incidence of sarcomas and other tumors of mesenchymal origin. This syndrome results from mutations in the WRN DNA helicase/exonuclease gene. We found that WRN deficient primary fibroblasts, as well as lymphoblastoid cell lines (LCLs), show reduced proliferative survival in response to 4-nitroquinoline-N-oxide (4NQO) and 8-methoxypsoralen (8MOP), compared with WRN-proficient cells. This is the first demonstration of drug hypersensitivity in primary cells of mesenchymal origin from WRN patients. Notably, 8MOP-induced DNA interstrand crosslinks, but not 8MOP mono-adducts, produced S-phase apoptosis in WRN-deficient LCLs. In contrast, 8MOP did not induce S-phase apoptosis in WRN-deficient diploid fibroblasts, in which drug hypersensitivity was entirely due to reduced cell proliferation. Such reduced proliferation of damaged mesenchymal cells in WRN patients may lead to earlier proliferative senescence. In addition, failure of WRN-deficient mesenchymal cells to undergo apoptosis in response to DNA damage in S-phase may promote genomic instability and could help clarify the increased risk of sarcoma in WRN patients. Because interstrand crosslinks are believed to be repaired through homologous recombination, these results suggest an important role for WRN in recombinational resolution of stalled replication forks. (+info)
Regulation of the Werner helicase through a direct interaction with a subunit of protein kinase A.
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Werner syndrome is a hereditary disease characterized by cancer predisposition, genetic instability, and the premature appearance of features associated with normal aging. At the molecular level this syndrome has been related to mutations in the Werner helicase, a member of the RecQ family of DNA helicases which are required to maintain genomic stability in cells. Here we show by a yeast two-hybrid screen that the Werner helicase can directly interact with the regulatory subunit (RIbeta) of cAMP protein kinase A (PKA). We confirm that this interaction occurs in vivo. Interestingly, serum withdrawal causes a redistribution of the Werner helicase within the nucleus of mammalian cells. Raising intracellular cAMP levels or increased expression of the regulatory but not the catalytic subunit of PKA inhibits this nuclear redistribution stimulated by serum deprivation. These results suggest that similar to lower organisms, gene products linked to genomic instability and aging may be directly regulated by growth factor-sensitive, PKA-dependent pathways. (+info)
Homologous recombination resolution defect in werner syndrome.
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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 hypersensitivity, 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. (+info)
Genetic interaction between the unstable v-Ha-RAS transgene (Tg.AC) and the murine Werner syndrome gene: transgene instability and tumorigenesis.
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Tg.AC transgenic mice provide a sensitive assay for oncogenic agents and a convenient alternative to the two-stage initiation/promoter model of skin tumorigenesis. Although extensively used, this model has remained in part an enigma since mice that carry the Tg.AC transgene (consisting of v-Ha-Ras driven by an embryonic zeta-globin promoter) would not ordinarily be expected to develop skin and other adult tumors. Cloning and characterizing the inserted transgene has provided an insight into the Tg.AC phenotype. We find that the transgene is inserted into a Line-1 element in such a way as to create extended inverted repeats consisting of both transgene and Line-1 sequences. Such structures would be expected to contribute to the instability of the Tg.AC locus and we suggest that this instability is critical to the Tg.AC phenotype. Further, we strengthen this notion by introducing an inactivating mutation in the murine Wrn gene (a gene important in maintenance of genome stability) and showing that bigenic Tg.AC/Wrn(Deltahel/Deltahel) mice experience an eightfold increase in inactivating germline mutations at the Tg.AC locus. Similarly, Tg.AC/Wrn(Deltahel/Deltahel) mice that retain an intact and thus active Tg.AC locus experience a sharp increase in papillomas as compared to Tg.AC/Wrn(+/+) mice. This work demonstrates a genetic interaction between the instability of the multicopy transgene and the Werner Syndrome gene. From this, we conclude that genetic instability remains a key element in this tumor promoter model. (+info)
A nucleolar targeting sequence in the Werner syndrome protein resides within residues 949-1092.
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Werner syndrome is a premature aging disorder caused by the lack of an active Werner syndrome protein (WRN). The patients suffer from many of the ailments seen at a much later stage in the life of normal individuals. WRN is a nuclear protein and contains a nuclear localization signal (NLS) in its C-terminal region. Inside the nucleus, WRN is mainly located in the nucleoli and in nuclear foci. To begin to understand the role of WRN in the nucleolus, we determined the specific regions of the protein that are responsible for this localization. We have cloned different WRN gene domains fused to enhanced green fluorescent protein (EGFP), and analyzed their intracellular distribution in living cells using confocal microscopy. The region encompassing amino acids 949-1092 of the human WRN, together with the NLS containing amino acids 1358-1432, provides the targeting to the nucleoli. This targeting is observed in three human and one mouse cell line. The NLS-containing region alone is unable to direct EGFP to the nucleoli. The results demonstrate that the human WRN contains a conserved nucleolar targeting sequence residing in a 144 amino acid region (aa 949-1092) and this provides new tools and insight into the biological function of WRN. (+info)
DNA damage-induced translocation of the Werner helicase is regulated by acetylation.
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Werner syndrome is a rare autosomal recessive disorder involving the premature appearance of features reminiscent of human aging. Werner syndrome occurs by mutation of the WRN gene, encoding a DNA helicase. WRN contributes to the induction of the p53 tumor suppressor protein by various DNA damaging agents. Here we show that UV exposure leads to extensive translocation of WRN from the nucleolus to nucleoplasmic foci in a dose-dependent manner. Ionizing radiation also induces WRN translocation, albeit milder, partially through activation of the ATM kinase. The nucleoplasmic foci to which WRN is recruited display partial colocalization with PML nuclear bodies. The translocation of WRN into nucleoplasmic foci is significantly enhanced by the protein deacetylase inhibitor, Trichostatin A. Moreover, Trichostatin A delays the re-entry of WRN into the nucleolus at late times after irradiation. WRN is acetylated in vivo, and this is markedly stimulated by the acetyltransferase p300. Importantly, p300 augments the translocation of WRN into nucleoplasmic foci. These findings support the notion that WRN plays a role in the cellular response to DNA damage and suggest that the activity of WRN is modulated by DNA damage-induced post-translational modifications of WRN and possibly WRN-interacting proteins. (+info)