Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Science 22 September 2000:
Vol. 289. no. 5487, pp. 2133 - 2137
DOI: 10.1126/science.289.5487.2133
Prev | Table of Contents | Next

Reports

Activation of the DNA Replication Checkpoint Through RNA Synthesis by Primase

W. Matthew Michael,12* Robert Ott,3 Ellen Fanning,3 John Newport1

When DNA replication is inhibited during the synthesis (S) phase of the cell cycle, a signaling pathway (checkpoint) is activated that serves to prevent mitosis from initiating before completion of replication. This replication checkpoint acts by down-regulating the activity of the mitotic inducer cdc2-cyclin B. Here, we report the relation between chromatin structure and induction of the replication checkpoint. Chromatin was competent to initiate a checkpoint response only after the DNA was unwound and DNA polymerase alpha  had been loaded. Checkpoint induction did not require new DNA synthesis on the unwound template strand but did require RNA primer synthesis by primase. These findings identify the RNA portion of the primer as an important component of the signal that activates the replication checkpoint.

1 Department of Biology, University of California, San Diego, La Jolla, CA 92093-0349, USA.
2 Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
3 Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235, USA.
*   To whom correspondence should be addressed. E-mail: matt{at}mcb.harvard.edu

Read the Full Text


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Timeless Maintains Genomic Stability and Suppresses Sister Chromatid Exchange during Unperturbed DNA Replication.
K. A. Urtishak, K. D. Smith, R. A. Chanoux, R. A. Greenberg, F. B. Johnson, and E. J. Brown (2009)
J. Biol. Chem. 284, 8777-8785
   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 »
Yeast telomere capping protein Stn1 overrides DNA replication control through the S phase checkpoint.
H. J. Gasparyan, L. Xu, R. C. Petreaca, A. E. Rex, V. Y. Small, N. S. Bhogal, J. A. Julius, T. H. Warsi, J. Bachant, O. M. Aparicio, et al. (2009)
PNAS 106, 2206-2211
   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 »
Recombinant Adeno-Associated Viral Vectors Are Deficient in Provoking a DNA Damage Response.
M. Fragkos, M. Breuleux, N. Clement, and P. Beard (2008)
J. Virol. 82, 7379-7387
   Abstract »    Full Text »    PDF »
Mutation of DNA primase causes extensive apoptosis of retinal neurons through the activation of DNA damage checkpoint and tumor suppressor p53.
M. Yamaguchi, N. Fujimori-Tonou, Y. Yoshimura, T. Kishi, H. Okamoto, and I. Masai (2008)
Development 135, 1247-1257
   Abstract »    Full Text »    PDF »
Orchestration of the S-phase and DNA damage checkpoint pathways by replication forks from early origins.
J. M. Caldwell, Y. Chen, K. L. Schollaert, J. F. Theis, G. F. Babcock, C. S. Newlon, and Y. Sanchez (2008)
J. Cell Biol. 180, 1073-1086
   Abstract »    Full Text »    PDF »
Domain Architecture and Biochemical Characterization of Vertebrate Mcm10.
P. D. Robertson, E. M. Warren, H. Zhang, D. B. Friedman, J. W. Lary, J. L. Cole, A. V. Tutter, J. C. Walter, E. Fanning, and B. F. Eichman (2008)
J. Biol. Chem. 283, 3338-3348
   Abstract »    Full Text »    PDF »
The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted.
E. Liu, A. Y.-L. Lee, T. Chiba, E. Olson, P. Sun, and X. Wu (2007)
J. Cell Biol. 179, 643-657
   Abstract »    Full Text »    PDF »
An Iron-Sulfur Cluster in the C-terminal Domain of the p58 Subunit of Human DNA Primase.
B. E. Weiner, H. Huang, B. M. Dattilo, M. J. Nilges, E. Fanning, and W. J. Chazin (2007)
J. Biol. Chem. 282, 33444-33451
   Abstract »    Full Text »    PDF »
Tipin is required for stalled replication forks to resume DNA replication after removal of aphidicolin in Xenopus egg extracts.
A. Errico, V. Costanzo, and T. Hunt (2007)
PNAS 104, 14929-14934
   Abstract »    Full Text »    PDF »
Reconstitution of a human ATR-mediated checkpoint response to damaged DNA.
J.-H. Choi, L. A. Lindsey-Boltz, and A. Sancar (2007)
PNAS 104, 13301-13306
   Abstract »    Full Text »    PDF »
The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1.
S. Delacroix, J. M. Wagner, M. Kobayashi, K.-i. Yamamoto, and L. M. Karnitz (2007)
Genes & Dev. 21, 1472-1477
   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
   Full Text »    PDF »
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
   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 »
Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling.
S. Yan, H. D. Lindsay, and W. M. Michael (2006)
J. Cell Biol. 173, 181-186
   Abstract »    Full Text »    PDF »
Protein Phosphatase 2A Antagonizes ATM and ATR in a Cdk2- and Cdc7-Independent DNA Damage Checkpoint..
P. Petersen, D. M. Chou, Z. You, T. Hunter, J. C. Walter, and G. Walter (2006)
Mol. Cell. Biol. 26, 1997-2011
   Abstract »    Full Text »    PDF »
Recruitment of DNA Damage Checkpoint Proteins to Damage in Transcribed and Nontranscribed Sequences.
G. Jiang and A. Sancar (2006)
Mol. Cell. Biol. 26, 39-49
   Abstract »    Full Text »    PDF »
Activation of the ATR Pathway by Human Immunodeficiency Virus Type 1 Vpr Involves Its Direct Binding to Chromatin In Vivo.
M. Lai, E. S. Zimmerman, V. Planelles, and J. Chen (2005)
J. Virol. 79, 15443-15451
   Abstract »    Full Text »    PDF »
Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus Egg Extracts Requires Binding of ATRIP to ATR but Not the Stable DNA-binding or Coiled-coil Domains of ATRIP.
S.-M. Kim, A. Kumagai, J. Lee, and W. G. Dunphy (2005)
J. Biol. Chem. 280, 38355-38364
   Abstract »    Full Text »    PDF »
Roles of Replication Fork-interacting and Chk1-activating Domains from Claspin in a DNA Replication Checkpoint Response.
J. Lee, D. A. Gold, A. Shevchenko, A. Shevchenko, and W. G. Dunphy (2005)
Mol. Biol. Cell 16, 5269-5282
   Abstract »    Full Text »    PDF »
Proliferating Cell Nuclear Antigen Recruits Cyclin-dependent Kinase Inhibitor Xic1 to DNA and Couples Its Proteolysis to DNA Polymerase Switching.
L.-C. Chuang and P. R. Yew (2005)
J. Biol. Chem. 280, 35299-35309
   Abstract »    Full Text »    PDF »
DNA Polymerase {kappa} Is Specifically Required for Recovery from the Benzo[a]pyrene-Dihydrodiol Epoxide (BPDE)-induced S-phase Checkpoint.
X. Bi, D. M. Slater, H. Ohmori, and C. Vaziri (2005)
J. Biol. Chem. 280, 22343-22355
   Abstract »    Full Text »    PDF »
Unwind and slow down: checkpoint activation by helicase and polymerase uncoupling.
D. Cortez (2005)
Genes & Dev. 19, 1007-1012
   Full Text »    PDF »
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 »
ATRIP Binding to Replication Protein A-Single-stranded DNA Promotes ATR-ATRIP Localization but Is Dispensable for Chk1 Phosphorylation.
H. L. Ball, J. S. Myers, and D. Cortez (2005)
Mol. Biol. Cell 16, 2372-2381
   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 »
Mcl1p Is a Polymerase {alpha} Replication Accessory Factor Important for S-Phase DNA Damage Survival.
D. R. Williams and J. R. McIntosh (2005)
Eukaryot. Cell 4, 166-177
   Abstract »    Full Text »    PDF »
Characterization of a novel ATR-dependent, Chk1-independent, intra-S-phase checkpoint that suppresses initiation of replication in Xenopus.
M. G. Luciani, M. Oehlmann, and J. J. Blow (2004)
J. Cell Sci. 117, 6019-6030
   Abstract »    Full Text »    PDF »
The DNA primase of Sulfolobus solfataricus is activated by substrates containing a thymine-rich bubble and has a 3'-terminal nucleotidyl-transferase activity.
M. De Falco, A. Fusco, M. De Felice, M. Rossi, and F. M. Pisani (2004)
Nucleic Acids Res. 32, 5223-5230
   Abstract »    Full Text »    PDF »
Replication protein A-mediated recruitment and activation of Rad17 complexes.
L. Zou, D. Liu, and S. J. Elledge (2003)
PNAS 100, 13827-13832
   Abstract »    Full Text »    PDF »
Phosphorylated Claspin Interacts with a Phosphate-binding Site in the Kinase Domain of Chk1 during ATR-mediated Activation.
S.-Y. Jeong, A. Kumagai, J. Lee, and W. G. Dunphy (2003)
J. Biol. Chem. 278, 46782-46788
   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 »
Regulation of Cdc2/Cyclin B Activation in Xenopus Egg Extracts via Inhibitory Phosphorylation of Cdc25C Phosphatase by Ca2+/Calmodium-dependent Kinase II.
J. R. A. Hutchins, D. Dikovskaya, and P. R. Clarke (2003)
Mol. Biol. Cell 14, 4003-4014
   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 »
Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes.
L. Zou and S. J. Elledge (2003)
Science 300, 1542-1548
   Abstract »    Full Text »    PDF »
Negative Regulation of Mitotic Promoting Factor by the Checkpoint Kinase Chk1 in Simian Virus 40 Lytic Infection.
E. Okubo, J. M. Lehman, and T. D. Friedrich (2002)
J. Virol. 77, 1257-1267
   Abstract »    Full Text »    PDF »
The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication.
R. A. Van Hatten, A. V. Tutter, A. H. Holway, A. M. Khederian, J. C. Walter, and W. M. Michael (2002)
J. Cell Biol. 159, 541-547
   Abstract »    Full Text »    PDF »
Geminin Deficiency Causes a Chk1-dependent G2 Arrest in Xenopus.
T. J. McGarry (2002)
Mol. Biol. Cell 13, 3662-3671
   Abstract »    Full Text »    PDF »
A requirement for replication in activation of the ATR-dependent DNA damage checkpoint.
P. J. Lupardus, T. Byun, M.-c. Yee, M. Hekmat-Nejad, and K. A. Cimprich (2002)
Genes & Dev. 16, 2327-2332
   Abstract »    Full Text »    PDF »
DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts.
M. P. Stokes, R. Van Hatten, H. D. Lindsay, and W. M. Michael (2002)
J. Cell Biol. 158, 863-872
   Abstract »    Full Text »    PDF »
MCM2-7 Complexes Bind Chromatin in a Distributed Pattern Surrounding the Origin Recognition Complex in Xenopus Egg Extracts.
M. C. Edwards, A. V. Tutter, C. Cvetic, C. H. Gilbert, T. A. Prokhorova, and J. C. Walter (2002)
J. Biol. Chem. 277, 33049-33057
   Abstract »    Full Text »    PDF »
The Role of Single-stranded DNA and Polymerase alpha in Establishing the ATR, Hus1 DNA Replication Checkpoint.
Z. You, L. Kong, and J. Newport (2002)
J. Biol. Chem. 277, 27088-27093
   Abstract »    Full Text »    PDF »
Targeted Destruction of DNA Replication Protein Cdc6 by Cell Death Pathways in Mammals and Yeast.
F. Blanchard, M. E. Rusiniak, K. Sharma, X. Sun, I. Todorov, M. M. Castellano, C. Gutierrez, H. Baumann, and W. C. Burhans (2002)
Mol. Biol. Cell 13, 1536-1549
   Abstract »    Full Text »    PDF »
An oxidized nucleotide affects DNA replication through activation of protein kinases in Xenopus egg lysates.
T. Kai, R. Matsunaga, M. Eguchi, H. Kamiya, H. Kasai, M. Suzuki, and S. Izuta (2002)
Nucleic Acids Res. 30, 569-573
   Abstract »    Full Text »    PDF »
MCM2-7 Proteins Are Essential Components of Prereplicative Complexes that Accumulate Cooperatively in the Nucleus during G1-phase and Are Required to Establish, But Not Maintain, the S-phase Checkpoint.
K. Labib, S. E. Kearsey, and J. F.X. Diffley (2001)
Mol. Biol. Cell 12, 3658-3667
   Abstract »    Full Text »    PDF »
The Drosophila Geminin homolog: roles for Geminin in limiting DNA replication, in anaphase and in neurogenesis.
L. M. Quinn, A. Herr, T. J. McGarry, and H. Richardson (2001)
Genes & Dev. 15, 2741-2754
   Abstract »    Full Text »    PDF »
Cell cycle checkpoint signaling through the ATM and ATR kinases.
R. T. Abraham (2001)
Genes & Dev. 15, 2177-2196
   Full Text »    PDF »
ATR inhibition selectively sensitizes G1 checkpoint-deficient cells to lethal premature chromatin condensation.
P. Nghiem, P. K. Park, Y.-s. Kim, C. Vaziri, and S. L. Schreiber (2001)
PNAS 98, 9092-9097
   Abstract »    Full Text »    PDF »
In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase {alpha}-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides.
J. D. Purviance, A. E. Prack, B. Barbaro, and P. A. Bullock (2001)
J. Virol. 75, 6392-6401
   Abstract »    Full Text »
S and G2 Phase Roles for Cdk2 Revealed by Inducible Expression of a Dominant-Negative Mutant in Human Cells.
B. Hu, J. Mitra, S. van den Heuvel, and G. H. Enders (2001)
Mol. Cell. Biol. 21, 2755-2766
   Abstract »    Full Text »
Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains.
A. Lengronne, P. Pasero, A. Bensimon, and E. Schwob (2001)
Nucleic Acids Res. 29, 1433-1442
   Abstract »    Full Text »    PDF »
DNA replication blockade impairs p53-transactivation.
R. Takimoto and W. S. El-Deiry (2001)
PNAS 98, 781-783
   Full Text »    PDF »


To Advertise     Find Products

Science. ISSN 0036-8075 (print), 1095-9203 (online)