|
|
|
|
|
||
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
Research Article |
1 Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences,
Tohoku University, Aoba-ku, Sendai, Miyagi 980-8578, Japan
2 Department of Biophysics and Biochemistry, Graduate School of Science,
University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
* Author for correspondence (e-mail: tada{at}mail.pharm.tohoku.ac.jp )
Accepted 12 May 2002
The response to DNA damage was analyzed using a cell-free system consisting
of Xenopus egg extract and demembranated sperm nuclei. In the absence
of DNA-damaging agents, detergent-resistant accumulation of replication
protein A appeared in nuclei after a 30 minute incubation, and a considerable
portion of the replication protein A signals disappeared during a further 30
minute incubation. Similar replication protein A accumulation was observed in
the nuclei after a 30 minute incubation in the extract containing
camptothecin, whereas a further 30 minute incubation generated discrete
replication protein A foci. The addition of camptothecin also induced
formation of 
-H2AX foci, which have been previously shown to localize
at sites of DSBs. Analysis of the time course of DNA replication and results
obtained using geminin, an inhibitor of licensing for DNA replication, suggest
that the discrete replication protein A foci formed in response to
camptothecin-induced DNA damage occur in a DNA-replication-dependent manner.
When the nuclei were incubated in the extract containing EcoRI,
discrete replication protein A foci were observed at 30 minutes as well as at
60 and 90 minutes after incubation, and the focus-formation of replication
protein A was not sensitive to geminin. DNA replication was almost completely
inhibited in the presence of EcoRI and the inhibition was sensitive
to caffeine, an inhibitor of ataxia telangiectasia mutated protein (ATM) and
ATM- and Rad3-related protein (ATR). However, the focus-formation of
replication protein A in the presence of EcoRI was not influenced by
caffeine treatment. EcoRI-induced incorporation of biotin-dUTP into
chromatin was observed following geminin-mediated inhibition of DNA
replication, suggesting that the incorporation was the result of DNA repair.
The biotin-dUTP signal co-localized with replication protein A foci and was
not significantly suppressed or stimulated by the addition of caffeine.
Key words: Double-strand DNA break, DNA repair, Checkpoint, Xenopus egg extract, Replication protein A, Camptothecin, EcoRI
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Y. Yoo, A. Kumagai, A. Shevchenko, A. Shevchenko, and W. G. Dunphy The Mre11-Rad50-Nbs1 Complex Mediates Activation of TopBP1 by ATM Mol. Biol. Cell, May 1, 2009; 20(9): 2351 - 2360. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Y. Yoo, A. Kumagai, A. Shevchenko, A. Shevchenko, and W. G. Dunphy Ataxia-telangiectasia Mutated (ATM)-dependent Activation of ATR Occurs through Phosphorylation of TopBP1 by ATM J. Biol. Chem., June 15, 2007; 282(24): 17501 - 17506. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Sasakawa, T. Fukui, and S. Waga Accumulation of FFA-1, the Xenopus Homolog of Werner Helicase, and DNA Polymerase {delta} on Chromatin in Response to Replication Fork Arrest J. Biochem., July 1, 2006; 140(1): 95 - 103. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Y. Yoo, S.-Y. Jeong, and W. G. Dunphy Site-specific phosphorylation of a checkpoint mediator protein controls its responses to different DNA structures Genes & Dev., April 1, 2006; 20(7): 772 - 783. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sobeck, S. Stone, V. Costanzo, B. de Graaf, T. Reuter, J. de Winter, M. Wallisch, Y. Akkari, S. Olson, W. Wang, et al. Fanconi Anemia Proteins Are Required To Prevent Accumulation of Replication-Associated DNA Double-Strand Breaks Mol. Cell. Biol., January 15, 2006; 26(2): 425 - 437. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Yan, J. McCane, T. Toczylowski, and C. Chen Analysis of the Xenopus Werner syndrome protein in DNA double-strand break repair J. Cell Biol., October 24, 2005; 171(2): 217 - 227. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Tsuyama, S. Tada, S. Watanabe, M. Seki, and T. Enomoto Licensing for DNA replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts Nucleic Acids Res., February 1, 2005; 33(2): 765 - 775. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Y. Yoo, A. Shevchenko, A. Shevchenko, and W. G. Dunphy Mcm2 Is a Direct Substrate of ATM and ATR during DNA Damage and DNA Replication Checkpoint Responses J. Biol. Chem., December 17, 2004; 279(51): 53353 - 53364. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Francon, J.-M. Lemaitre, C. Dreyer, D. Maiorano, O. Cuvier, and M. Mechali A hypophosphorylated form of RPA34 is a specific component of pre-replication centers J. Cell Sci., October 1, 2004; 117(21): 4909 - 4920. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Markey, H. Siddiqui, and E. S. Knudsen Geminin Is Targeted for Repression by the Retinoblastoma Tumor Suppressor Pathway through Intragenic E2F Sites J. Biol. Chem., July 9, 2004; 279(28): 29255 - 29262. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Li, S.-M. Kim, J. Lee, and W. G. Dunphy Absence of BLM leads to accumulation of chromosomal DNA breaks during both unperturbed and disrupted S phases J. Cell Biol., June 21, 2004; 165(6): 801 - 812. [Abstract] [Full Text] [PDF]
|
|
