Yan H, McCane J, Toczylowski T, Chen C.

R01 GM57962-02 NIGMS NIH HHS , R01 GM057962 NIGMS NIH HHS

	Figure 1. xWRN is recruited to DSB foci. Top: DSBs induce the formation of discrete foci that contain xWRN and RPA. Nuclei were reconstituted in cytosol and membrane fractions in the presence of NcoI (+NcoI; 0.25 unit/μl) or buffer (−NcoI). After 60 min, nuclei were fixed and costained with affinity-purified rabbit anti-xWRN and rat anti-RPA followed by goat anti–rabbit FITC and goat anti–rat Texas Red.
	Figure 2. Depletion of xWRN does not affect NHEJ. (A) Depletion of xWRN from cytosol. XWRN- or mock-depleted cytosol was loaded on a 7% SDS-PAGE, transferred to an Immobilon P membrane, and probed with the purified rabbit anti-xWRN antibodies. The two lanes on the right are quantitation controls and contain normal cytosol at 1% and 3% of the amount loaded in the lanes containing the depleted cytosol. (B) Linear pUC19 molecules (5 ng/μl for -B/P and 10 ng/μl for -B/H and -K/H) with different ends were incubated in xWRN-depleted or mock-depleted cytosol at room temperature for 2 h. Samples were treated with SDS/proteinase K, and separated on a 1% agarose gel. Substrates: pUC19-B/P: pUC19 digested by BamHI and PstI; pUC19-B/H: pUC19 digested by BamHI and HincII; pUC19-K/H: pUC19 digested by KpnI and HincII. −W: xWRN-depleted; −M: mock-depleted. (C) Junction sequences of the repaired products. The predicted sequences of perfectly repaired junctions are listed at the top.
	Figure 3. Establishment of SSA in NPE. (A) Preparation of the SSA substrate pRW4′. Plasmid pRW4 was digested with XhoI and then partially filled in by TTP and dCTP with Klenow (exo-; NEB, NE). (B) pRW4′ (12 ng/μl) was incubated in NPE at room temperature. Samples were taken at the indicated times, treated with SDS/proteinase K, and separated on a 1% agarose gel. Lanes 1–4: time points of the reaction in NPE; lane 5: XhoI-digested pRW4 ligated with T4 DNA ligase; lane 6: uncut pRW4; lane 7: pRW4′; lane 8: pRW4′ ligated with T4 DNA ligase. Bands indicated by (*) are NHEJ products. (C) Restriction digestion of the 10-kb repair product (indicated by the line in B). Left: predicted digestion pattern by SalI and EcoRI; middle and right: gel electrophoresis of the digested DNA. The faint bands above the 4.36 band are due to partial digestion. (D) Restriction digestion of the cloned EcoRI fragment. Left: gel electrophoresis of the digested plasmid; right: predicted digestion patterns of the pBR322 plasmid and pRW4 plasmid. X: XhoI site. (E) Gel electrophoresis of the junction DNA directly amplified from the 10-kb repair product.
	Figure 4. Distinction between SSA and HR. (A) Two potential pathways for generating the 10-kb repair product. SSA uses ss-tail for annealing with the complementary ss-tail, whereas HR uses the ss-tail for invasion of ds-DNA to form a D-loop. (B) Substrates used to distinguish between SSA and HR. Digestion by ClaI and XbaI generates pACYC-c/x and places the Tet gene at the end. Digestion by NcoI generates pACYC-n and places the Tet gene in the middle. (C) Gel electrophoresis of the repair products after DNA substrates (12 ng/μl each) were incubated in NPE at room temperature for the indicated times. All three DNA substrates were extended with TTP, dATP, dCTP, and ddGTP. pRW4′′ is different from pRW4′ in that it contains an extra ddGTP at the end. Bands indicated by (*) are hybrid repair products formed between pRW4′′ and pACYC-cx.
	Figure 5. Effect of xRAD51. (A) Western blot of the xRAD51- or mock-depleted NPE. The four lanes on the right are quantitation controls and contain normal NPE at 10%, 5%, 2%, and 1% of the amount loaded in the depleted NPE. (B) SSA assay with xRAD51-depleted and mock-depleted NPE. The substrate (pRW4′) was incubated in NPE for the indicated times, treated with SDS/proteinase K, and separated by agarose gel electrophoresis. The SSA products include the band indicated by the arrow and a subset of the bands indicated by the bracket.
	Figure 6. Depletion of xWRN reduces SSA. (A) Western blot of the depleted NPE. The three lanes on the right are quantitation controls and contain normal NPE at 1%, 2%, and 10% of the amount loaded in the lanes containing the depleted NPE. (B) SSA assay with xWRN-depleted and mock-depleted NPE. pRW4′ was incubated in NPE for the indicated times, treated with SDS/proteinase K, and separated by agarose gel electrophoresis. The SSA products include the band indicated by the arrow and a subset of the bands indicated by the bracket. (C) Staining intensity plot of the lanes containing the 2-h repair products in (B).
	Figure 7. Rescue of SSA by the xWRN protein. (A) Silver staining of the xWRN protein purified from Xenopus egg cytosol by conventional column chromatography (Yan et al., 1998). (B) Add-back of purified xWRN to the xWRN-depleted NPE. PRW4′ was incubated in xWRN-depleted NPE supplemented with xWRN (5 ng/μl final concentration) or buffer (ELB) for the indicated times and then analyzed by agarose gel electrophoresis. The SSA products include the band indicated by the arrow and a subset of the bands in the bracketed area. (C) Staining intensity plot of the lanes containing the 3-h repair products in (B).

Baumann, Role of the human RAD51 protein in homologous recombination and double-stranded-break repair. 1998, Pubmed

Baumann, Role of the human RAD51 protein in homologous recombination and double-stranded-break repair. 1998, Pubmed
Blander, DNA damage-induced translocation of the Werner helicase is regulated by acetylation. 2002, Pubmed
Brosh, Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity. 2001, Pubmed
Carroll, Homologous genetic recombination in Xenopus: mechanism and implications for gene manipulation. 1996, Pubmed , Xenbase
Chen, Evidence for a replication function of FFA-1, the Xenopus orthologue of Werner syndrome protein. 2001, Pubmed , Xenbase
Comai, The Werner syndrome protein at the crossroads of DNA repair and apoptosis. 2004, Pubmed
Constantinou, Werner's syndrome protein (WRN) migrates Holliday junctions and co-localizes with RPA upon replication arrest. 2000, Pubmed
Cooper, Ku complex interacts with and stimulates the Werner protein. 2000, Pubmed
Cox, The importance of repairing stalled replication forks. 2000, Pubmed
Crabbe, Defective telomere lagging strand synthesis in cells lacking WRN helicase activity. 2004, Pubmed
Fujiwara, A retarded rate of DNA replication and normal level of DNA repair in Werner's syndrome fibroblasts in culture. 1977, Pubmed
Fujiwara, Abnormal fibroblast aging and DNA replication in the Werner syndrome. 1985, Pubmed
Fukuchi, Mutator phenotype of Werner syndrome is characterized by extensive deletions. 1989, Pubmed
Fukuchi, Increased frequency of 6-thioguanine-resistant peripheral blood lymphocytes in Werner syndrome patients. 1990, Pubmed
Grandi, DNA double-strand breaks induce formation of RP-A/Ku foci on in vitro reconstituted Xenopus sperm nuclei. 2001, Pubmed , Xenbase
Gray, Werner helicase is localized to transcriptionally active nucleoli of cycling cells. 1998, Pubmed
Hanaoka, Decrease in the average size of replicons in a Werner syndrome cell line by Simian virus 40 infection. 1983, Pubmed
Hoehn, Variegated translocation mosaicism in human skin fibroblast cultures. 1975, Pubmed
Ivanov, Genetic requirements for the single-strand annealing pathway of double-strand break repair in Saccharomyces cerevisiae. 1996, Pubmed
Kamath-Loeb, Functional interaction between the Werner Syndrome protein and DNA polymerase delta. 2000, Pubmed
Khakhar, RecQ helicases: multiple roles in genome maintenance. 2003, Pubmed
Kobayashi, Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract. 2002, Pubmed , Xenbase
Labhart, Ku-dependent nonhomologous DNA end joining in Xenopus egg extracts. 1999, Pubmed , Xenbase
Lander, Initial sequencing and analysis of the human genome. 2001, Pubmed
Larminat, Deficiency in BRCA2 leads to increase in non-conservative homologous recombination. 2002, Pubmed
Lebel, The Werner syndrome gene product co-purifies with the DNA replication complex and interacts with PCNA and topoisomerase I. 1999, Pubmed
Lebel, A deletion within the murine Werner syndrome helicase induces sensitivity to inhibitors of topoisomerase and loss of cellular proliferative capacity. 1998, Pubmed
Lee, Pathways and functions of the Werner syndrome protein. 2005, Pubmed
Li, Functional interaction between Ku and the werner syndrome protein in DNA end processing. 2000, Pubmed
Liang, Homology-directed repair is a major double-strand break repair pathway in mammalian cells. 1998, Pubmed
Lisby, Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. 2004, Pubmed
Lohka, Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. 1983, Pubmed , Xenbase
Lopes, The DNA replication checkpoint response stabilizes stalled replication forks. 2001, Pubmed
Martin, Replicative life-span of cultivated human cells. Effects of donor's age, tissue, and genotype. 1970, Pubmed
Maryon, Degradation of linear DNA by a strand-specific exonuclease activity in Xenopus laevis oocytes. 1989, Pubmed , Xenbase
McGlynn, Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled. 2001, Pubmed
Mimura, Xenopus Cdc45-dependent loading of DNA polymerase alpha onto chromatin under the control of S-phase Cdk. 1998, Pubmed , Xenbase
Mushegian, Positionally cloned human disease genes: patterns of evolutionary conservation and functional motifs. 1997, Pubmed
Newport, Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. 1987, Pubmed , Xenbase
Orren, A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA. 2001, Pubmed
Oshima, Lack of WRN results in extensive deletion at nonhomologous joining ends. 2002, Pubmed
Pâques, Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae. 1997, Pubmed
Pfeiffer, Joining of nonhomologous DNA double strand breaks in vitro. 1988, Pubmed , Xenbase
Postow, Positive torsional strain causes the formation of a four-way junction at replication forks. 2001, Pubmed
Prince, Loss of Werner syndrome protein function promotes aberrant mitotic recombination. 2001, Pubmed
Rodríguez-López, Asymmetry of DNA replication fork progression in Werner's syndrome. 2002, Pubmed
Sakamoto, Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51. 2001, Pubmed
Salk, Cytogenetics of Werner's syndrome cultured skin fibroblasts: variegated translocation mosaicism. 1981, Pubmed
Scappaticci, Clonal structural chromosomal rearrangements in primary fibroblast cultures and in lymphocytes of patients with Werner's Syndrome. 1982, Pubmed
Shen, The Werner syndrome gene: the molecular basis of RecQ helicase-deficiency diseases. 2000, Pubmed
Smythe, Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts. 1991, Pubmed , Xenbase
Sugawara, Heteroduplex rejection during single-strand annealing requires Sgs1 helicase and mismatch repair proteins Msh2 and Msh6 but not Pms1. 2004, Pubmed
Sugawara, DNA length dependence of the single-strand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair. 2000, Pubmed
Symington, Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. 2002, Pubmed
Takeuchi, Prolongation of S phase and whole cell cycle in Werner's syndrome fibroblasts. 1982, Pubmed
Takeuchi, Altered frequency of initiation sites of DNA replication in Werner's syndrome cells. 1982, Pubmed
Thode, A novel pathway of DNA end-to-end joining. 1990, Pubmed , Xenbase
Tutt, Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences. 2001, Pubmed
Vilenchik, Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer. 2003, Pubmed , Xenbase
Walter, Regulated chromosomal DNA replication in the absence of a nucleus. 1998, Pubmed , Xenbase
Walter, Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha. 2000, Pubmed , Xenbase
Wu, Rules of donor preference in saccharomyces mating-type gene switching revealed by a competition assay involving two types of recombination. 1997, Pubmed
Yan, Replication focus-forming activity 1 and the Werner syndrome gene product. 1998, Pubmed , Xenbase
Yan, An analysis of the regulation of DNA synthesis by cdk2, Cip1, and licensing factor. 1995, Pubmed , Xenbase
Yan, Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast. 1993, Pubmed
Yu, Positional cloning of the Werner's syndrome gene. 1996, Pubmed