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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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Related in: MedlinePlus

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.
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fig5: 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.

Mentions: The second experiment that we did to distinguish SSA and HR was to examine RAD51 dependence. RAD51 is the mediator protein for strand invasion and is essential for D-loop formation during HR. SSA does not involve strand invasion, and genetic studies in Saccharomyces cerevisiae showed that it is independent of RAD51 (Ivanov et al., 1996). We depleted xRAD51 from NPE with purified anti-xRAD51 antibodies to at least >99% completion (Fig. 5 A). The xRAD51- and mock-depleted NPEs were incubated with linear pRW4 (pRW4′) as described in Fig. 3, and the repair products were separated by agarose gel electrophoresis. As shown in Fig. 5 B, there was no significant difference in the formation of repair products between the xRAD51-depleted and mock-depleted NPEs. Together, this result (independence of RAD51) and the previous result (dependence on homology at ends) strongly suggest that the homology-based repair in NPE is SSA rather than HR.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171202&req=5

fig5: 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.
Mentions: The second experiment that we did to distinguish SSA and HR was to examine RAD51 dependence. RAD51 is the mediator protein for strand invasion and is essential for D-loop formation during HR. SSA does not involve strand invasion, and genetic studies in Saccharomyces cerevisiae showed that it is independent of RAD51 (Ivanov et al., 1996). We depleted xRAD51 from NPE with purified anti-xRAD51 antibodies to at least >99% completion (Fig. 5 A). The xRAD51- and mock-depleted NPEs were incubated with linear pRW4 (pRW4′) as described in Fig. 3, and the repair products were separated by agarose gel electrophoresis. As shown in Fig. 5 B, there was no significant difference in the formation of repair products between the xRAD51-depleted and mock-depleted NPEs. Together, this result (independence of RAD51) and the previous result (dependence on homology at ends) strongly suggest that the homology-based repair in NPE is SSA rather than HR.

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