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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

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).
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fig6: 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).

Mentions: With the establishment of the SSA assay, we proceeded to determine if xWRN is important for this DSB repair pathway. xWRN was depleted from NPE with anti-xWRN antibodies to an undetectable level as judged by Western blot analysis (>98%) (Fig. 6 A). The depleted NPE was incubated with linear pRW4 (pRW4′) as described in Fig. 3, and the repair products were separated by agarose gel electrophoresis. As shown in Fig. 6 B, the SSA products (indicated by arrow and bracket) were reduced significantly in xWRN-depleted NPE. Quantification of band intensity showed that the residual SSA products were <10% of those in the mock-depleted NPE. In contrast to the SSA products, the NHEJ products were not reduced, but increased slightly. The experiment in Fig. 2 showed that xWRN depletion did not affect NHEJ. A possible simple explanation for the increase in NHEJ in this experiment is that the ends were no longer channeled into SSA, and thus more of them were available for NHEJ. The differential effect on SSA and NHEJ indicated that the extract was not inactivated nonspecifically by the depletion procedure.


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)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171202&req=5

fig6: 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).
Mentions: With the establishment of the SSA assay, we proceeded to determine if xWRN is important for this DSB repair pathway. xWRN was depleted from NPE with anti-xWRN antibodies to an undetectable level as judged by Western blot analysis (>98%) (Fig. 6 A). The depleted NPE was incubated with linear pRW4 (pRW4′) as described in Fig. 3, and the repair products were separated by agarose gel electrophoresis. As shown in Fig. 6 B, the SSA products (indicated by arrow and bracket) were reduced significantly in xWRN-depleted NPE. Quantification of band intensity showed that the residual SSA products were <10% of those in the mock-depleted NPE. In contrast to the SSA products, the NHEJ products were not reduced, but increased slightly. The experiment in Fig. 2 showed that xWRN depletion did not affect NHEJ. A possible simple explanation for the increase in NHEJ in this experiment is that the ends were no longer channeled into SSA, and thus more of them were available for NHEJ. The differential effect on SSA and NHEJ indicated that the extract was not inactivated nonspecifically by the depletion procedure.

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