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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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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.
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fig2: 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.

Mentions: We then investigated if xWRN is important for the NHEJ pathway of DSB repair. Xenopus egg extracts contain robust NHEJ activity that can repair a variety of DNA ends (Pfeiffer and Vielmetter, 1988; Labhart, 1999). We depleted xWRN from cytosol to an undetectable level (>97%; Fig. 2 A) following a procedure that was described before (Chen et al., 2001). The depleted cytosol was incubated with linear pUC19 DNA carrying three different combinations of ends: 5′/blunt (BamHI/HincII), 3′/blunt (KpnI/HincII), and 5′/3′ (BamHI/PstI). As shown in Fig. 2 B, repair products corresponding to supercoiled (I) and nicked circular (II) pUC19, as well as linear dimers and trimers, were formed readily after incubation. Compared with mock depletion, xWRN depletion had no significant effect on the formation of these products.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171202&req=5

fig2: 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.
Mentions: We then investigated if xWRN is important for the NHEJ pathway of DSB repair. Xenopus egg extracts contain robust NHEJ activity that can repair a variety of DNA ends (Pfeiffer and Vielmetter, 1988; Labhart, 1999). We depleted xWRN from cytosol to an undetectable level (>97%; Fig. 2 A) following a procedure that was described before (Chen et al., 2001). The depleted cytosol was incubated with linear pUC19 DNA carrying three different combinations of ends: 5′/blunt (BamHI/HincII), 3′/blunt (KpnI/HincII), and 5′/3′ (BamHI/PstI). As shown in Fig. 2 B, repair products corresponding to supercoiled (I) and nicked circular (II) pUC19, as well as linear dimers and trimers, were formed readily after incubation. Compared with mock depletion, xWRN depletion had no significant effect on the formation of these products.

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