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J. Cell Biol. 158 (5): 863-872

Copyright © 2002 by the Rockefeller University Press.


DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts

Matthew P. Stokes1, Ruth Van Hatten1, Howard D. Lindsay2, and W. Matthew Michael1

1 The Biological Laboratories, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
2 Genome Damage and Stability Center, University of Sussex Falmer, Brighton BN1 9QG, UK

Address correspondence to W. Matthew Michael, The Biological Laboratories, Dept. of Molecular and Cellular Biology, 16 Divinity Ave., Harvard University, Cambridge, MA 02138. Tel.: (617) 496-2940. Fax: (617) 384-7423. E-mail: matt{at}

Abstract: Alkylating agents, such as methyl methanesulfonate (MMS), damage DNA and activate the DNA damage checkpoint. Although many of the checkpoint proteins that transduce damage signals have been identified and characterized, the mechanism that senses the damage and activates the checkpoint is not yet understood. To address this issue for alkylation damage, we have reconstituted the checkpoint response to MMS in Xenopus egg extracts. Using four different indicators for checkpoint activation (delay on entrance into mitosis, slowing of DNA replication, phosphorylation of the Chk1 protein, and physical association of the Rad17 checkpoint protein with damaged DNA), we report that MMS-induced checkpoint activation is dependent upon entrance into S phase. Additionally, we show that the replication of damaged double-stranded DNA, and not replication of damaged single-stranded DNA, is the molecular event that activates the checkpoint. Therefore, these data provide direct evidence that replication forks are an obligate intermediate in the activation of the DNA damage checkpoint.

Key Words: cell cycle; Rad17; DNA damage; DNA replication; S phase

Control of DNA Replication by the Nucleus/Cytoplasm Ratio in Xenopus.
C. M. Murphy and W. M. Michael (2013)
J. Biol. Chem. 288, 29382-29393
   Abstract »    Full Text »    PDF »
Depletion of Uhrf1 inhibits chromosomal DNA replication in Xenopus egg extracts.
E. M. Taylor, N. M. Bonsu, R. J. Price, and H. D. Lindsay (2013)
Nucleic Acids Res. 41, 7725-7737
   Abstract »    Full Text »    PDF »
Role of replication protein A as sensor in activation of the S-phase checkpoint in Xenopus egg extracts.
B. Recolin, S. Van Der Laan, and D. Maiorano (2012)
Nucleic Acids Res. 40, 3431-3442
   Abstract »    Full Text »    PDF »
Safeguarding genome integrity: the checkpoint kinases ATR, CHK1 and WEE1 restrain CDK activity during normal DNA replication.
C. S. Sorensen and R. G. Syljuasen (2012)
Nucleic Acids Res. 40, 477-486
   Abstract »    Full Text »    PDF »
DNA is a co-factor for its own replication in Xenopus egg extracts.
R. Lebofsky, A. M. van Oijen, and J. C. Walter (2011)
Nucleic Acids Res. 39, 545-555
   Abstract »    Full Text »    PDF »
ATR activation and replication fork restart are defective in FANCM-deficient cells.
R. A. Schwab, A. N. Blackford, and W. Niedzwiedz (2010)
EMBO J. 29, 806-818
   Abstract »    Full Text »    PDF »
XRCC1 interacts with the p58 subunit of DNA Pol {alpha}-primase and may coordinate DNA repair and replication during S phase.
N. Levy, M. Oehlmann, F. Delalande, H. P. Nasheuer, A. Van Dorsselaer, V. Schreiber, G. de Murcia, J. Menissier-de Murcia, D. Maiorano, and A. Bresson (2009)
Nucleic Acids Res. 37, 3177-3188
   Abstract »    Full Text »    PDF »
TopBP1 and DNA polymerase-{alpha} directly recruit the 9-1-1 complex to stalled DNA replication forks.
S. Yan and W. M. Michael (2009)
J. Cell Biol. 184, 793-804
   Abstract »    Full Text »    PDF »
The Basic Cleft of RPA70N Binds Multiple Checkpoint Proteins, Including RAD9, To Regulate ATR Signaling.
X. Xu, S. Vaithiyalingam, G. G. Glick, D. A. Mordes, W. J. Chazin, and D. Cortez (2008)
Mol. Cell. Biol. 28, 7345-7353
   Abstract »    Full Text »    PDF »
Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response.
L. Zou (2007)
Genes & Dev. 21, 879-885
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The structural determinants of checkpoint activation.
C. A. MacDougall, T. S. Byun, C. Van, M.-c. Yee, and K. A. Cimprich (2007)
Genes & Dev. 21, 898-903
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Monoubiquitination of Proliferating Cell Nuclear Antigen Induced by Stalled Replication Requires Uncoupling of DNA Polymerase and Mini-chromosome Maintenance Helicase Activities.
D. J. Chang, P. J. Lupardus, and K. A. Cimprich (2006)
J. Biol. Chem. 281, 32081-32088
   Abstract »    Full Text »    PDF »
Chk1- and Claspin-Dependent but ATR/ATM- and Rad17-Independent DNA Replication Checkpoint Response in HeLa Cells..
V. Rodriguez-Bravo, S. Guaita-Esteruelas, R. Florensa, O. Bachs, and N. Agell (2006)
Cancer Res. 66, 8672-8679
   Abstract »    Full Text »    PDF »
Site-specific phosphorylation of a checkpoint mediator protein controls its responses to different DNA structures.
H. Y. Yoo, S.-Y. Jeong, and W. G. Dunphy (2006)
Genes & Dev. 20, 772-783
   Abstract »    Full Text »    PDF »
Phosphorylation of Xenopus Rad1 and Hus1 Defines a Readout for ATR Activation That Is Independent of Claspin and the Rad9 Carboxy Terminus.
P. J. Lupardus and K. A. Cimprich (2006)
Mol. Biol. Cell 17, 1559-1569
   Abstract »    Full Text »    PDF »
Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos.
A. H. Holway, S.-H. Kim, A. La Volpe, and W. M. Michael (2006)
J. Cell Biol. 172, 999-1008
   Abstract »    Full Text »    PDF »
Fanconi Anemia Proteins Are Required To Prevent Accumulation of Replication-Associated DNA Double-Strand Breaks.
A. Sobeck, S. Stone, V. Costanzo, B. de Graaf, T. Reuter, J. de Winter, M. Wallisch, Y. Akkari, S. Olson, W. Wang, et al. (2006)
Mol. Cell. Biol. 26, 425-437
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Unwind and slow down: checkpoint activation by helicase and polymerase uncoupling.
D. Cortez (2005)
Genes & Dev. 19, 1007-1012
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Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.
T. S. Byun, M. Pacek, M.-c. Yee, J. C. Walter, and K. A. Cimprich (2005)
Genes & Dev. 19, 1040-1052
   Abstract »    Full Text »    PDF »
G2 damage checkpoints: what is the turn-on?.
M. J. O'Connell and K. A. Cimprich (2005)
J. Cell Sci. 118, 1-6
   Abstract »    Full Text »    PDF »
A Tel1/MRX-Dependent Checkpoint Inhibits the Metaphase-to-Anaphase Transition after UV Irradiation in the Absence of Mec1.
M. Clerici, V. Baldo, D. Mantiero, F. Lottersberger, G. Lucchini, and M. P. Longhese (2004)
Mol. Cell. Biol. 24, 10126-10144
   Abstract »    Full Text »    PDF »
Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling.
C.-C. Tsao, C. Geisen, and R. T. Abraham (2004)
EMBO J. 23, 4660-4669
   Abstract »    Full Text »    PDF »
Xenopus Cds1 Is Regulated by DNA-Dependent Protein Kinase and ATR during the Cell Cycle Checkpoint Response to Double-Stranded DNA Ends.
T. D. McSherry and P. R. Mueller (2004)
Mol. Cell. Biol. 24, 9968-9985
   Abstract »    Full Text »    PDF »
Absence of BLM leads to accumulation of chromosomal DNA breaks during both unperturbed and disrupted S phases.
W. Li, S.-M. Kim, J. Lee, and W. G. Dunphy (2004)
J. Cell Biol. 165, 801-812
   Abstract »    Full Text »    PDF »
Visualization of Altered Replication Dynamics after DNA Damage in Human Cells.
C. J. Merrick, D. Jackson, and J. F. X. Diffley (2004)
J. Biol. Chem. 279, 20067-20075
   Abstract »    Full Text »    PDF »
A Novel Protein Activity Mediates DNA Binding of an ATR-ATRIP Complex.
R. D. Bomgarden, D. Yean, M.-C. Yee, and K. A. Cimprich (2004)
J. Biol. Chem. 279, 13346-13353
   Abstract »    Full Text »    PDF »
Cut5 Is Required for the Binding of Atr and DNA Polymerase {alpha} to Genotoxin-damaged Chromatin.
E. R. Parrilla-Castellar and L. M. Karnitz (2003)
J. Biol. Chem. 278, 45507-45511
   Abstract »    Full Text »    PDF »
DNA damage-induced replication arrest in Xenopus egg extracts.
M. P. Stokes and W. M. Michael (2003)
J. Cell Biol. 163, 245-255
   Abstract »    Full Text »    PDF »
The human checkpoint Rad protein Rad17 is chromatin-associated throughout the cell cycle, localizes to DNA replication sites, and interacts with DNA polymerase {epsilon}.
S. M. Post, A. E. Tomkinson, and E. Y.-H. P. Lee (2003)
Nucleic Acids Res. 31, 5568-5575
   Abstract »    Full Text »    PDF »
XRad17 Is Required for the Activation of XChk1 But Not XCds1 during Checkpoint Signaling in Xenopus.
R. E. Jones, J. R. Chapman, C. Puligilla, J. M. Murray, A. M. Car, C. C. Ford, and H. D. Lindsay (2003)
Mol. Biol. Cell 14, 3898-3910
   Abstract »    Full Text »    PDF »

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