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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

xWRN is recruited to DSB foci. Top: DSBs induce the formation of discrete foci that contain xWRN and RPA. Nuclei were reconstituted in cytosol and membrane fractions in the presence of NcoI (+NcoI; 0.25 unit/μl) or buffer (−NcoI). After 60 min, nuclei were fixed and costained with affinity-purified rabbit anti-xWRN and rat anti-RPA followed by goat anti–rabbit FITC and goat anti–rat Texas Red.
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fig1: xWRN is recruited to DSB foci. Top: DSBs induce the formation of discrete foci that contain xWRN and RPA. Nuclei were reconstituted in cytosol and membrane fractions in the presence of NcoI (+NcoI; 0.25 unit/μl) or buffer (−NcoI). After 60 min, nuclei were fixed and costained with affinity-purified rabbit anti-xWRN and rat anti-RPA followed by goat anti–rabbit FITC and goat anti–rat Texas Red.

Mentions: Many proteins that are involved in DSB repair are recruited to discrete subnuclear foci at the site of damage (Lisby et al., 2004). It was reported that human WRN is recruited to discrete foci in cells that are treated with various DNA damaging agents, but the agents that were used caused a variety of damages and it is unclear what type of DNA lesion actually triggers the formation of WRN foci (Sakamoto et al., 2001; Blander et al., 2002). To determine definitively if xWRN is recruited to DSB foci, we used restriction enzymes to introduce DSBs into chromatin. In brief, nuclei were reconstituted in the presence of NcoI (0.25 unit/μl) or buffer, incubated for 60 min, and then fixed and stained with antibodies. As shown in Fig. 1 (top panel), in the presence of NcoI, a large number of discrete foci containing xWRN and the eukaryotic single-strand DNA binding protein RPA were formed in the nuclei. Consistent with observations by other investigators (Grandi et al., 2001; Kobayashi et al., 2002), there is very little DNA synthesis in these nuclei because of the activation of checkpoint response that blocks DNA replication (unpublished data). In contrast, in the absence of NcoI, xWRN and RPA have a more granular staining pattern (Fig. 1, bottom panel). As reported earlier, this pattern coincides with extensive DNA replication at this time (Chen et al., 2001). In addition to NcoI, which generates 5′-protruding ends, restriction enzymes that generate 3′-protruding ends (KpnI) or blunt ends (StuI) were effective in inducing the formation of DSB foci (unpublished data). RPA was shown to be localized to sequences near DNA ends (Grandi et al., 2001), and physically interacts with xWRN and stimulates its helicase activity (Chen et al., 2001). These observations suggest that xWRN is part of foci formed at DNA ends. Additional staining with antibodies against replication initiation protein CDC45 (Mimura and Takisawa, 1998; Walter and Newport, 2000) and double-strand DNA end binding protein Ku (Labhart, 1999) showed that DSB foci are distinct from replication foci that are formed in normally reconstituted nuclei. Whereas DSB foci contain Ku, but not CDC45, replication foci contain CDC45, but not Ku (unpublished data). Together these observations are consistent with the notion that xWRN plays dual roles in DSB repair and replication.


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)

xWRN is recruited to DSB foci. Top: DSBs induce the formation of discrete foci that contain xWRN and RPA. Nuclei were reconstituted in cytosol and membrane fractions in the presence of NcoI (+NcoI; 0.25 unit/μl) or buffer (−NcoI). After 60 min, nuclei were fixed and costained with affinity-purified rabbit anti-xWRN and rat anti-RPA followed by goat anti–rabbit FITC and goat anti–rat Texas Red.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: xWRN is recruited to DSB foci. Top: DSBs induce the formation of discrete foci that contain xWRN and RPA. Nuclei were reconstituted in cytosol and membrane fractions in the presence of NcoI (+NcoI; 0.25 unit/μl) or buffer (−NcoI). After 60 min, nuclei were fixed and costained with affinity-purified rabbit anti-xWRN and rat anti-RPA followed by goat anti–rabbit FITC and goat anti–rat Texas Red.
Mentions: Many proteins that are involved in DSB repair are recruited to discrete subnuclear foci at the site of damage (Lisby et al., 2004). It was reported that human WRN is recruited to discrete foci in cells that are treated with various DNA damaging agents, but the agents that were used caused a variety of damages and it is unclear what type of DNA lesion actually triggers the formation of WRN foci (Sakamoto et al., 2001; Blander et al., 2002). To determine definitively if xWRN is recruited to DSB foci, we used restriction enzymes to introduce DSBs into chromatin. In brief, nuclei were reconstituted in the presence of NcoI (0.25 unit/μl) or buffer, incubated for 60 min, and then fixed and stained with antibodies. As shown in Fig. 1 (top panel), in the presence of NcoI, a large number of discrete foci containing xWRN and the eukaryotic single-strand DNA binding protein RPA were formed in the nuclei. Consistent with observations by other investigators (Grandi et al., 2001; Kobayashi et al., 2002), there is very little DNA synthesis in these nuclei because of the activation of checkpoint response that blocks DNA replication (unpublished data). In contrast, in the absence of NcoI, xWRN and RPA have a more granular staining pattern (Fig. 1, bottom panel). As reported earlier, this pattern coincides with extensive DNA replication at this time (Chen et al., 2001). In addition to NcoI, which generates 5′-protruding ends, restriction enzymes that generate 3′-protruding ends (KpnI) or blunt ends (StuI) were effective in inducing the formation of DSB foci (unpublished data). RPA was shown to be localized to sequences near DNA ends (Grandi et al., 2001), and physically interacts with xWRN and stimulates its helicase activity (Chen et al., 2001). These observations suggest that xWRN is part of foci formed at DNA ends. Additional staining with antibodies against replication initiation protein CDC45 (Mimura and Takisawa, 1998; Walter and Newport, 2000) and double-strand DNA end binding protein Ku (Labhart, 1999) showed that DSB foci are distinct from replication foci that are formed in normally reconstituted nuclei. Whereas DSB foci contain Ku, but not CDC45, replication foci contain CDC45, but not Ku (unpublished data). Together these observations are consistent with the notion that xWRN plays dual roles in DSB repair and replication.

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