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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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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).
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fig7: 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).

Mentions: To determine if the inhibitory effect of xWRN depletion on SSA was specific, the xWRN protein that was purified from Xenopus cytosol (Fig. 7 A) was added back to the xWRN-depleted NPE. The purification procedure for xWRN involved multiple types of chromatography resins, but not anti-xWRN antibodies (Yan et al., 1998). As shown in Fig. 7, B and C, the addition of this purified xWRN led to a significant rescue of SSA. These results strongly suggest that xWRN is important for the SSA pathway of DSB repair.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

fig7: 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).
Mentions: To determine if the inhibitory effect of xWRN depletion on SSA was specific, the xWRN protein that was purified from Xenopus cytosol (Fig. 7 A) was added back to the xWRN-depleted NPE. The purification procedure for xWRN involved multiple types of chromatography resins, but not anti-xWRN antibodies (Yan et al., 1998). As shown in Fig. 7, B and C, the addition of this purified xWRN led to a significant rescue of SSA. These results strongly suggest that xWRN is important for the SSA pathway of DSB repair.

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