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.

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.

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.

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).

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.

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.

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).