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Analysis of the Xenopus Werner syndrome protein in DNA double-strand break repair.

Yan H, McCane J, Toczylowski T, Chen C - J. Cell Biol. (2005)

Bottom Line: Werner syndrome is associated with premature aging and increased risk of cancer.Using Xenopus egg extracts as the model system, we found that Xenopus WRN (xWRN) is recruited to discrete foci upon induction of DSBs.Depletion of xWRN has no significant effect on nonhomologous end-joining of DSB ends, but it causes a significant reduction in the homology-dependent single-strand annealing DSB repair pathway.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, PA 19111, USA. Hong_Yan@fccc.edu

ABSTRACT
Werner syndrome is associated with premature aging and increased risk of cancer. Werner syndrome protein (WRN) is a RecQ-type DNA helicase, which seems to participate in DNA replication, double-strand break (DSB) repair, and telomere maintenance; however, its exact function remains elusive. Using Xenopus egg extracts as the model system, we found that Xenopus WRN (xWRN) is recruited to discrete foci upon induction of DSBs. Depletion of xWRN has no significant effect on nonhomologous end-joining of DSB ends, but it causes a significant reduction in the homology-dependent single-strand annealing DSB repair pathway. These results provide the first direct biochemical evidence that links WRN to a specific DSB repair pathway. The assay for single-strand annealing that was developed in this study also provides a powerful biochemical system for mechanistic analysis of homology-dependent DSB repair.

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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.
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fig3: 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.

Mentions: DSBs also can be repaired by two homology-dependent pathways: HR and SSA. Our attempt to establish an HR assay in Xenopus egg extracts was unsuccessful, but we did succeed in establishing a robust SSA assay. Previously, Carroll (1996) demonstrated that SSA can occur after DNA is injected into oocytes or unfertilized eggs or after it is incubated in germinal vesicle extracts. However, the injection method is incompatible with immunodepletion, and germinal vesicle extracts seem to be too dilute to survive immunodepletion. The assay that we established uses NPE, which is derived from nuclei reconstituted in Xenopus egg extracts (Walter et al., 1998). NPE contains high concentrations of nuclear proteins that can catalyze the replication of plasmid DNA. Similarly, we observed that NPE (but not total egg extracts nor membrane-free cytosol) contains robust activity for SSA. The DNA substrate for SSA is a 5.6-kb linearized plasmid, pRW4′ (with the XhoI ends partially filled in by dCTP and TTP to prevent simple religation), which carries two 1.2-kb direct repeats (tetracycline resistance gene; Tet) at the two ends (Fig. 3 A) (Maryon and Carroll, 1989). As shown in Fig. 3 B (lanes 1–4), after incubation in NPE, pRW4′ was converted into multiple products. Three of the bands (*) correspond to supercoiled circular monomer, nicked circular monomer, and linear dimer of pRW4′. They were not formed by simple religation, because the ends are not complementary (compare with lane 8). In addition to these three bands, we detected a prominent 10-kb band (and many higher molecular weight bands that were produced even when NHEJ was inhibited [see Fig. 4 C]). The size of the 10-kb band was consistent with the expected size of an intermolecular SSA reaction product. When this band was isolated from gel, its restriction digestion pattern was that expected of the SSA product (Fig. 3 C). The junction DNA was cloned by ligating the EcoRI-digested DNA with T4 ligase, followed by transformation into E. coli. The transformants were resistant to ampicillin and—when restreaked—to tetracycline (16/16; DNA not treated with T4 DNA ligase did not give rise to transformants). The plasmids that were isolated from the transformants were analyzed by restriction digestion and were found to have the same pattern as that of pBR322, the parent plasmid of pRW4′ (6/6; two shown in Fig. 3 D). Sequence analysis confirmed that the junction sequence in the clones is the same as the sequence around the Tet gene in pBR322 (Fig. S1; available at http://www.jcb.org/cgi/content/full/jcb.200502077/DC1). Furthermore, PCR amplification of the 10-kb repair product with two primers that bracket the Tet repeat gave rise to a 1.5-kb product as predicted from SSA (Fig. 3 E). Direct sequencing of this PCR product showed that it also is the same as the Tet gene in pBR322 (Fig. S2). Of particular importance is that the XhoI site between the two Tet repeats in pRW4 is missing in the cloned junction and the PCR-amplified junction (Figs. S1 and S2). Taken together, these results strongly suggest that the 10-kb DNA is composed of two linear pRW4 molecules linked in tandem, but with only one Tet repeat retained in between.


Analysis of the Xenopus Werner syndrome protein in DNA double-strand break repair.

Yan H, McCane J, Toczylowski T, Chen C - J. Cell Biol. (2005)

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.
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Related In: Results  -  Collection

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fig3: 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.
Mentions: DSBs also can be repaired by two homology-dependent pathways: HR and SSA. Our attempt to establish an HR assay in Xenopus egg extracts was unsuccessful, but we did succeed in establishing a robust SSA assay. Previously, Carroll (1996) demonstrated that SSA can occur after DNA is injected into oocytes or unfertilized eggs or after it is incubated in germinal vesicle extracts. However, the injection method is incompatible with immunodepletion, and germinal vesicle extracts seem to be too dilute to survive immunodepletion. The assay that we established uses NPE, which is derived from nuclei reconstituted in Xenopus egg extracts (Walter et al., 1998). NPE contains high concentrations of nuclear proteins that can catalyze the replication of plasmid DNA. Similarly, we observed that NPE (but not total egg extracts nor membrane-free cytosol) contains robust activity for SSA. The DNA substrate for SSA is a 5.6-kb linearized plasmid, pRW4′ (with the XhoI ends partially filled in by dCTP and TTP to prevent simple religation), which carries two 1.2-kb direct repeats (tetracycline resistance gene; Tet) at the two ends (Fig. 3 A) (Maryon and Carroll, 1989). As shown in Fig. 3 B (lanes 1–4), after incubation in NPE, pRW4′ was converted into multiple products. Three of the bands (*) correspond to supercoiled circular monomer, nicked circular monomer, and linear dimer of pRW4′. They were not formed by simple religation, because the ends are not complementary (compare with lane 8). In addition to these three bands, we detected a prominent 10-kb band (and many higher molecular weight bands that were produced even when NHEJ was inhibited [see Fig. 4 C]). The size of the 10-kb band was consistent with the expected size of an intermolecular SSA reaction product. When this band was isolated from gel, its restriction digestion pattern was that expected of the SSA product (Fig. 3 C). The junction DNA was cloned by ligating the EcoRI-digested DNA with T4 ligase, followed by transformation into E. coli. The transformants were resistant to ampicillin and—when restreaked—to tetracycline (16/16; DNA not treated with T4 DNA ligase did not give rise to transformants). The plasmids that were isolated from the transformants were analyzed by restriction digestion and were found to have the same pattern as that of pBR322, the parent plasmid of pRW4′ (6/6; two shown in Fig. 3 D). Sequence analysis confirmed that the junction sequence in the clones is the same as the sequence around the Tet gene in pBR322 (Fig. S1; available at http://www.jcb.org/cgi/content/full/jcb.200502077/DC1). Furthermore, PCR amplification of the 10-kb repair product with two primers that bracket the Tet repeat gave rise to a 1.5-kb product as predicted from SSA (Fig. 3 E). Direct sequencing of this PCR product showed that it also is the same as the Tet gene in pBR322 (Fig. S2). Of particular importance is that the XhoI site between the two Tet repeats in pRW4 is missing in the cloned junction and the PCR-amplified junction (Figs. S1 and S2). Taken together, these results strongly suggest that the 10-kb DNA is composed of two linear pRW4 molecules linked in tandem, but with only one Tet repeat retained in between.

Bottom Line: Werner syndrome is associated with premature aging and increased risk of cancer.Using Xenopus egg extracts as the model system, we found that Xenopus WRN (xWRN) is recruited to discrete foci upon induction of DSBs.Depletion of xWRN has no significant effect on nonhomologous end-joining of DSB ends, but it causes a significant reduction in the homology-dependent single-strand annealing DSB repair pathway.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, PA 19111, USA. Hong_Yan@fccc.edu

ABSTRACT
Werner syndrome is associated with premature aging and increased risk of cancer. Werner syndrome protein (WRN) is a RecQ-type DNA helicase, which seems to participate in DNA replication, double-strand break (DSB) repair, and telomere maintenance; however, its exact function remains elusive. Using Xenopus egg extracts as the model system, we found that Xenopus WRN (xWRN) is recruited to discrete foci upon induction of DSBs. Depletion of xWRN has no significant effect on nonhomologous end-joining of DSB ends, but it causes a significant reduction in the homology-dependent single-strand annealing DSB repair pathway. These results provide the first direct biochemical evidence that links WRN to a specific DSB repair pathway. The assay for single-strand annealing that was developed in this study also provides a powerful biochemical system for mechanistic analysis of homology-dependent DSB repair.

Show MeSH
Related in: MedlinePlus