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.
Subscribe

Logo for

Science 297 (5581): 599-602

Copyright © 2002 by the American Association for the Advancement of Science

Fork Reversal and ssDNA Accumulation at Stalled Replication Forks Owing to Checkpoint Defects

José M. Sogo,1* Massimo Lopes,2* Marco Foiani2dagger

Checkpoint-mediated control of replicating chromosomes is essential for preventing cancer. In yeast, Rad53 kinase protects stalled replication forks from pathological rearrangements. To characterize the mechanisms controlling fork integrity, we analyzed replication intermediates formed in response to replication blocks using electron microscopy. At the forks, wild-type cells accumulate short single-stranded regions, which likely causes checkpoint activation, whereas rad53 mutants exhibit extensive single-stranded gaps and hemi-replicated intermediates, consistent with a lagging-strand synthesis defect. Further, rad53 cells accumulate Holliday junctions through fork reversal. We speculate that, in checkpoint mutants, abnormal replication intermediates begin to form because of uncoordinated replication and are further processed by unscheduled recombination pathways, causing genome instability.

1 Institute of Cell Biology, ETH Hönggerberg, CH-8093 Zürich, Switzerland.
2 Istituto F.I.R.C. di Oncologia Molecolare, Via Adamello 16, 20141, Milano, Italy, and Dipartimento di Genetica e di Biologia dei Microrganismi, Università degli Studi di Milano, Via Celoria 26, 20133, Milano, Italy.
*   These authors contributed equally to this work.

dagger    To whom correspondence should be addressed. E-mail: foiani{at}ifom-firc.it

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Rescuing Stalled or Damaged Replication Forks.
J. T. P. Yeeles, J. Poli, K. J. Marians, and P. Pasero (2013)
Cold Spring Harb Perspect Biol 5, a012815
   Abstract »    Full Text »    PDF »
Back to the Origin: Reconsidering Replication, Transcription, Epigenetics, and Cell Cycle Control.
A. Evertts and H. A. Coller (2013)
Genes & Cancer
   Abstract »    Full Text »    PDF »
FANCJ couples replication past natural fork barriers with maintenance of chromatin structure.
R. A. Schwab, J. Nieminuszczy, K. Shin-ya, and W. Niedzwiedz (2013)
J. Cell Biol. 201, 33-48
   Abstract »    Full Text »    PDF »
Oncogenes induce genotoxic stress by mitotic processing of unusual replication intermediates.
K. J. Neelsen, I. M. Y. Zanini, R. Herrador, and M. Lopes (2013)
J. Cell Biol. 200, 699-708
   Abstract »    Full Text »    PDF »
Cdc45 Protein-Single-stranded DNA Interaction Is Important for Stalling the Helicase during Replication Stress.
I. Bruck and D. L. Kaplan (2013)
J. Biol. Chem. 288, 7550-7563
   Abstract »    Full Text »    PDF »
Mature Microsatellites: Mechanisms Underlying Dinucleotide Microsatellite Mutational Biases in Human Cells.
B. A. Baptiste, G. Ananda, N. Strubczewski, A. Lutzkanin, S. J. Khoo, A. Srikanth, N. Kim, K. D. Makova, M. M. Krasilnikova, and K. A. Eckert (2013)
g3 3, 451-463
   Abstract »    Full Text »    PDF »
Oligodeoxynucleotide Binding to (CTG) {middle dot} (CAG) Microsatellite Repeats Inhibits Replication Fork Stalling, Hairpin Formation, and Genome Instability.
G. Liu, X. Chen, and M. Leffak (2013)
Mol. Cell. Biol. 33, 571-581
   Abstract »    Full Text »    PDF »
The Saccharomyces cerevisiae F-Box Protein Dia2 Is a Mediator of S-Phase Checkpoint Recovery from DNA Damage.
C. M. Fong, A. Arumugam, and D. M. Koepp (2013)
Genetics 193, 483-499
   Abstract »    Full Text »    PDF »
G1/S and G2/M Cyclin-Dependent Kinase Activities Commit Cells to Death in the Absence of the S-Phase Checkpoint.
N. Manfrini, E. Gobbini, V. Baldo, C. Trovesi, G. Lucchini, and M. P. Longhese (2012)
Mol. Cell. Biol. 32, 4971-4985
   Abstract »    Full Text »    PDF »
Continued DNA Synthesis in Replication Checkpoint Mutants Leads to Fork Collapse.
S. A. Sabatinos, M. D. Green, and S. L. Forsburg (2012)
Mol. Cell. Biol. 32, 4986-4997
   Abstract »    Full Text »    PDF »
Genome Rearrangements Caused by Depletion of Essential DNA Replication Proteins in Saccharomyces cerevisiae.
E. Cheng, J. A. Vaisica, J. Ou, A. Baryshnikova, Y. Lu, F. P. Roth, and G. W. Brown (2012)
Genetics 192, 147-160
   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 »
Human DNA Helicase B (HDHB) Binds to Replication Protein A and Facilitates Cellular Recovery from Replication Stress.
G. D. Guler, H. Liu, S. Vaithiyalingam, D. R. Arnett, E. Kremmer, W. J. Chazin, and E. Fanning (2012)
J. Biol. Chem. 287, 6469-6481
   Abstract »    Full Text »    PDF »
Functional Analysis of DNA Replication Fork Reversal Catalyzed by Mycobacterium tuberculosis RuvAB Proteins.
J. S. Khanduja and K. Muniyappa (2012)
J. Biol. Chem. 287, 1345-1360
   Abstract »    Full Text »    PDF »
Mms1 and Mms22 stabilize the replisome during replication stress.
J. A. Vaisica, A. Baryshnikova, M. Costanzo, C. Boone, and G. W. Brown (2011)
Mol. Biol. Cell 22, 2396-2408
   Abstract »    Full Text »    PDF »
Quantitative Proteomic Analysis of Chromatin Reveals that Ctf18 Acts in the DNA Replication Checkpoint.
T. Kubota, S.-i. Hiraga, K. Yamada, A. I. Lamond, and A. D. Donaldson (2011)
Mol. Cell. Proteomics 10, M110.005561
   Abstract »    Full Text »    PDF »
The Arabidopsis thaliana Checkpoint Kinase WEE1 Protects against Premature Vascular Differentiation during Replication Stress.
T. Cools, A. Iantcheva, A. K. Weimer, S. Boens, N. Takahashi, S. Maes, H. Van den Daele, G. Van Isterdael, A. Schnittger, and L. De Veylder (2011)
PLANT CELL 23, 1435-1448
   Abstract »    Full Text »    PDF »
Regulation of fragile sites expression in budding yeast by MEC1, RRM3 and hydroxyurea.
N. Hashash, A. L. Johnson, and R. S. Cha (2011)
J. Cell Sci. 124, 181-185
   Abstract »    Full Text »    PDF »
The Transcription Elongation Factor Bur1-Bur2 Interacts with Replication Protein A and Maintains Genome Stability during Replication Stress.
E. Clausing, A. Mayer, S. Chanarat, B. Muller, S. M. Germann, P. Cramer, M. Lisby, and K. Strasser (2010)
J. Biol. Chem. 285, 41665-41674
   Abstract »    Full Text »    PDF »
Checkpoint genes and Exo1 regulate nearby inverted repeat fusions that form dicentric chromosomes in Saccharomyces cerevisiae.
S. Kaochar, L. Shanks, and T. Weinert (2010)
PNAS 107, 21605-21610
   Abstract »    Full Text »    PDF »
Cyclin Regulation by the S Phase Checkpoint.
G. Palou, R. Palou, A. Guerra-Moreno, A. Duch, A. Travesa, and D. G. Quintana (2010)
J. Biol. Chem. 285, 26431-26440
   Abstract »    Full Text »    PDF »
Continued primer synthesis at stalled replication forks contributes to checkpoint activation.
C. Van, S. Yan, W. M. Michael, S. Waga, and K. A. Cimprich (2010)
J. Cell Biol. 189, 233-246
   Abstract »    Full Text »    PDF »
Elevated dNTP levels suppress hyper-recombination in Saccharomyces cerevisiae S-phase checkpoint mutants.
M. Fasullo, O. Tsaponina, M. Sun, and A. Chabes (2010)
Nucleic Acids Res. 38, 1195-1203
   Abstract »    Full Text »    PDF »
Alternative Mechanisms for Coordinating Polymerase {alpha} and MCM Helicase.
C. Lee, I. Liachko, R. Bouten, Z. Kelman, and B. K. Tye (2010)
Mol. Cell. Biol. 30, 423-435
   Abstract »    Full Text »    PDF »
Leaping forks at inverted repeats.
D. Branzei and M. Foiani (2010)
Genes & Dev. 24, 5-9
   Abstract »    Full Text »    PDF »
Identification of SMARCAL1 as a Component of the DNA Damage Response.
L. Postow, E. M. Woo, B. T. Chait, and H. Funabiki (2009)
J. Biol. Chem. 284, 35951-35961
   Abstract »    Full Text »    PDF »
The Mcm Complex: Unwinding the Mechanism of a Replicative Helicase.
M. L. Bochman and A. Schwacha (2009)
Microbiol. Mol. Biol. Rev. 73, 652-683
   Abstract »    Full Text »    PDF »
Centromere Replication Timing Determines Different Forms of Genomic Instability in Saccharomyces cerevisiae Checkpoint Mutants During Replication Stress.
W. Feng, J. Bachant, D. Collingwood, M. K. Raghuraman, and B. J. Brewer (2009)
Genetics 183, 1249-1260
   Abstract »    Full Text »    PDF »
Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress.
V. M. Vassin, R. W. Anantha, E. Sokolova, S. Kanner, and J. A. Borowiec (2009)
J. Cell Sci. 122, 4070-4080
   Abstract »    Full Text »    PDF »
Mammalian Rif1 contributes to replication stress survival and homology-directed repair.
S. B.C. Buonomo, Y. Wu, D. Ferguson, and T. de Lange (2009)
J. Cell Biol. 187, 385-398
   Abstract »    Full Text »    PDF »
Arabidopsis Replication Protein A 70a is Required for DNA Damage Response and Telomere Length Homeostasis.
Y. Takashi, Y. Kobayashi, K. Tanaka, and K. Tamura (2009)
Plant Cell Physiol. 50, 1965-1976
   Abstract »    Full Text »    PDF »
HARPing on about the DNA damage response during replication.
R. Driscoll and K. A. Cimprich (2009)
Genes & Dev. 23, 2359-2365
   Abstract »    Full Text »    PDF »
Replication Stress Leads to Genome Instabilities in Arabidopsis DNA Polymerase {delta} Mutants.
D. Schuermann, O. Fritsch, J. M. Lucht, and B. Hohn (2009)
PLANT CELL 21, 2700-2714
   Abstract »    Full Text »    PDF »
Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo.
R. C. Burgess, M. Lisby, V. Altmannova, L. Krejci, P. Sung, and R. Rothstein (2009)
J. Cell Biol. 185, 969-981
   Abstract »    Full Text »    PDF »
Role of DNA damage-induced replication checkpoint in promoting lesion bypass by translesion synthesis in yeast.
V. Pages, S. R. Santa Maria, L. Prakash, and S. Prakash (2009)
Genes & Dev. 23, 1438-1449
   Abstract »    Full Text »    PDF »
Replication fork reversal and the maintenance of genome stability.
J. Atkinson and P. McGlynn (2009)
Nucleic Acids Res. 37, 3475-3492
   Abstract »    Full Text »    PDF »
Schizosaccharomyces pombe Rtf2 mediates site-specific replication termination by inhibiting replication restart.
T. Inagawa, T. Yamada-Inagawa, T. Eydmann, I. S. Mian, T. S. Wang, and J. Z. Dalgaard (2009)
PNAS 106, 7927-7932
   Abstract »    Full Text »    PDF »
Polymerase Dynamics at the Eukaryotic DNA Replication Fork.
P. M. J. Burgers (2009)
J. Biol. Chem. 284, 4041-4045
   Abstract »    Full Text »    PDF »
Fragile Genomic Sites Are Associated with Origins of Replication.
S. C. Di Rienzi, D. Collingwood, M. K. Raghuraman, and B. J. Brewer (2009)
Genome Biol Evol 1, 350-363
   Abstract »    Full Text »    PDF »
Remodeling of DNA replication structures by the branch point translocase FANCM.
K. Gari, C. Decaillet, M. Delannoy, L. Wu, and A. Constantinou (2008)
PNAS 105, 16107-16112
   Abstract »    Full Text »    PDF »
Activity of Hydroxyurea against Leishmania mexicana.
H. Martinez-Rojano, J. Mancilla-Ramirez, L. Quinonez-Diaz, and N. Galindo-Sevilla (2008)
Antimicrob. Agents Chemother. 52, 3642-3647
   Abstract »    Full Text »    PDF »
Mcm4 C-terminal domain of MCM helicase prevents excessive formation of single-stranded DNA at stalled replication forks.
N. Nitani, C. Yadani, H. Yabuuchi, H. Masukata, and T. Nakagawa (2008)
PNAS 105, 12973-12978
   Abstract »    Full Text »    PDF »
Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae.
S. J. Szyjka, J. G. Aparicio, C. J. Viggiani, S. Knott, W. Xu, S. Tavare, and O. M. Aparicio (2008)
Genes & Dev. 22, 1906-1920
   Abstract »    Full Text »    PDF »
Separate roles for the DNA damage checkpoint protein kinases in stabilizing DNA replication forks.
M. Segurado and J. F.X. Diffley (2008)
Genes & Dev. 22, 1816-1827
   Abstract »    Full Text »    PDF »
Distinct Phosphatases Mediate the Deactivation of the DNA Damage Checkpoint Kinase Rad53.
A. Travesa, A. Duch, and D. G. Quintana (2008)
J. Biol. Chem. 283, 17123-17130
   Abstract »    Full Text »    PDF »
Hjm/Hel308A DNA Helicase from Sulfolobus tokodaii Promotes Replication Fork Regression and Interacts with Hjc Endonuclease In Vitro.
Z. Li, S. Lu, G. Hou, X. Ma, D. Sheng, J. Ni, and Y. Shen (2008)
J. Bacteriol. 190, 3006-3017
   Abstract »    Full Text »    PDF »
Loss of CHK1 function impedes DNA damage-induced FANCD2 monoubiquitination but normalizes the abnormal G2 arrest in Fanconi anemia.
J.-H. Guervilly, G. Mace-Aime, and F. Rosselli (2008)
Hum. Mol. Genet. 17, 679-689
   Abstract »    Full Text »    PDF »
Minichromosome Maintenance Proteins Interact with Checkpoint and Recombination Proteins To Promote S-Phase Genome Stability.
J. M. Bailis, D. D. Luche, T. Hunter, and S. L. Forsburg (2008)
Mol. Cell. Biol. 28, 1724-1738
   Abstract »    Full Text »    PDF »
ATR-dependent pathways control hEXO1 stability in response to stalled forks.
M. El-Shemerly, D. Hess, A. K. Pyakurel, S. Moselhy, and S. Ferrari (2008)
Nucleic Acids Res. 36, 511-519
   Abstract »    Full Text »    PDF »
RFCCtf18 and the Swi1-Swi3 Complex Function in Separate and Redundant Pathways Required for the Stabilization of Replication Forks to Facilitate Sister Chromatid Cohesion in Schizosaccharomyces pombe.
A. B. Ansbach, C. Noguchi, I. W. Klansek, M. Heidlebaugh, T. M. Nakamura, and E. Noguchi (2008)
Mol. Biol. Cell 19, 595-607
   Abstract »    Full Text »    PDF »
Cleavage of Stalled Forks by Fission Yeast Mus81/Eme1 in Absence of DNA Replication Checkpoint.
B. Froget, J. Blaisonneau, S. Lambert, and G. Baldacci (2008)
Mol. Biol. Cell 19, 445-456
   Abstract »    Full Text »    PDF »
The processing of double-strand breaks and binding of single-strand-binding proteins RPA and Rad51 modulate the formation of ATR-kinase foci in yeast.
K. Dubrana, H. van Attikum, F. Hediger, and S. M. Gasser (2007)
J. Cell Sci. 120, 4209-4220
   Abstract »    Full Text »    PDF »
Identification of Mutations That Decrease the Stability of a Fragment of Saccharomyces cerevisiae Chromosome III Lacking Efficient Replicators.
J. F. Theis, A. Dershowitz, C. Irene, C. Maciariello, M. L. Tobin, G. Liberi, S. Tabrizifard, M. Korus, L. Fabiani, and C. S. Newlon (2007)
Genetics 177, 1445-1458
   Abstract »    Full Text »    PDF »
Replication in Hydroxyurea: It's a Matter of Time.
G. M. Alvino, D. Collingwood, J. M. Murphy, J. Delrow, B. J. Brewer, and M. K. Raghuraman (2007)
Mol. Cell. Biol. 27, 6396-6406
   Abstract »    Full Text »    PDF »
Replication Fork Reversal Occurs Spontaneously after Digestion but Is Constrained in Supercoiled Domains.
M. Fierro-Fernandez, P. Hernandez, D. B. Krimer, and J. B. Schvartzman (2007)
J. Biol. Chem. 282, 18190-18196
   Abstract »    Full Text »    PDF »
Cooperative Roles of Vertebrate Fbh1 and Blm DNA Helicases in Avoidance of Crossovers during Recombination Initiated by Replication Fork Collapse.
M. Kohzaki, A. Hatanaka, E. Sonoda, M. Yamazoe, K. Kikuchi, N. Vu Trung, D. Szuts, J. E. Sale, H. Shinagawa, M. Watanabe, et al. (2007)
Mol. Cell. Biol. 27, 2812-2820
   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 »
Anaphase Onset Before Complete DNA Replication with Intact Checkpoint Responses.
J. Torres-Rosell, G. De Piccoli, V. Cordon-Preciado, S. Farmer, A. Jarmuz, F. Machin, P. Pasero, M. Lisby, J. E. Haber, and L. Aragon (2007)
Science 315, 1411-1415
   Abstract »    Full Text »    PDF »
Replication Fork Stalling at Natural Impediments.
E. V. Mirkin and S. M. Mirkin (2007)
Microbiol. Mol. Biol. Rev. 71, 13-35
   Abstract »    Full Text »    PDF »
Increased Common Fragile Site Expression, Cell Proliferation Defects, and Apoptosis following Conditional Inactivation of Mouse Hus1 in Primary Cultured Cells.
M. Zhu and R. S. Weiss (2007)
Mol. Biol. Cell 18, 1044-1055
   Abstract »    Full Text »    PDF »
Sap1 Promotes the Association of the Replication Fork Protection Complex With Chromatin and Is Involved in the Replication Checkpoint in Schizosaccharomyces pombe.
C. Noguchi and E. Noguchi (2007)
Genetics 175, 553-566
   Abstract »    Full Text »    PDF »
Topological locking restrains replication fork reversal.
M. Fierro-Fernandez, P. Hernandez, D. B. Krimer, A. Stasiak, and J. B. Schvartzman (2007)
PNAS 104, 1500-1505
   Abstract »    Full Text »    PDF »
Ascorbate acts as a highly potent inducer of chromate mutagenesis and clastogenesis: linkage to DNA breaks in G2 phase by mismatch repair.
M. Reynolds, L. Stoddard, I. Bespalov, and A. Zhitkovich (2007)
Nucleic Acids Res. 35, 465-476
   Abstract »    Full Text »    PDF »
Ccr4 contributes to tolerance of replication stress through control of CRT1 mRNA poly(A) tail length.
R. N. Woolstencroft, T. H. Beilharz, M. A. Cook, T. Preiss, D. Durocher, and M. Tyers (2006)
J. Cell Sci. 119, 5178-5192
   Abstract »    Full Text »    PDF »
The F-Box Protein Dia2 Overcomes Replication Impedance to Promote Genome Stability in Saccharomyces cerevisiae.
D. Blake, B. Luke, P. Kanellis, P. Jorgensen, T. Goh, S. Penfold, B.-J. Breitkreutz, D. Durocher, M. Peter, and M. Tyers (2006)
Genetics 174, 1709-1727
   Abstract »    Full Text »    PDF »
The budding yeast protein Chl1p is required to preserve genome integrity upon DNA damage in S-phase.
S. Laha, S. P. Das, S. Hajra, S. Sau, and P. Sinha (2006)
Nucleic Acids Res. 34, 5880-5891
   Abstract »    Full Text »    PDF »
Limiting amounts of budding yeast Rad53 S-phase checkpoint activity results in increased resistance to DNA alkylation damage.
V. Cordon-Preciado, S. Ufano, and A. Bueno (2006)
Nucleic Acids Res. 34, 5852-5862
   Abstract »    Full Text »    PDF »
Human RECQ5{beta} helicase promotes strand exchange on synthetic DNA structures resembling a stalled replication fork.
R. Kanagaraj, N. Saydam, P. L. Garcia, L. Zheng, and P. Janscak (2006)
Nucleic Acids Res.
   Abstract »    Full Text »    PDF »
Coordination of DNA synthesis and replicative unwinding by the S-phase checkpoint pathways.
M. N. Nedelcheva-Veleva, D. B. Krastev, and S. S. Stoynov (2006)
Nucleic Acids Res. 34, 4138-4146
   Abstract »    Full Text »    PDF »
RecQ helicases: lessons from model organisms.
J. A. Cobb and L. Bjergbaek (2006)
Nucleic Acids Res. 34, 4106-4114
   Abstract »    Full Text »    PDF »
Novel Role for Checkpoint Rad53 Protein Kinase in the Initiation of Chromosomal DNA Replication in Saccharomyces cerevisiae.
P. R. Dohrmann and R. A. Sclafani (2006)
Genetics 174, 87-99
   Abstract »    Full Text »    PDF »
Regulation of DNA Replication Machinery by Mrc1 in Fission Yeast.
N. Nitani, K.-i. Nakamura, C. Nakagawa, H. Masukata, and T. Nakagawa (2006)
Genetics 174, 155-165
   Abstract »    Full Text »    PDF »
Promyelocytic leukemia nuclear bodies are predetermined processing sites for damaged DNA.
S. O. Boe, M. Haave, A. Jul-Larsen, A. Grudic, R. Bjerkvig, and P. E. Lonning (2006)
J. Cell Sci. 119, 3284-3295
   Abstract »    Full Text »    PDF »
The Bloom's Syndrome Helicase Can Promote the Regression of a Model Replication Fork.
C. Ralf, I. D. Hickson, and L. Wu (2006)
J. Biol. Chem. 281, 22839-22846
   Abstract »    Full Text »    PDF »
A Conserved Hsp10-like Domain in Mcm10 Is Required to Stabilize the Catalytic Subunit of DNA Polymerase-{alpha} in Budding Yeast.
R. M. Ricke and A.-K. Bielinsky (2006)
J. Biol. Chem. 281, 18414-18425
   Abstract »    Full Text »    PDF »
DNA damage responses and their many interactions with the replication fork.
P. R. Andreassen, G. P.H. Ho, and A. D. D'Andrea (2006)
Carcinogenesis 27, 883-892
   Abstract »    Full Text »    PDF »
Smc5p Promotes Faithful Chromosome Transmission and DNA Repair in Saccharomyces cerevisiae.
G. J. Cost and N. R. Cozzarelli (2006)
Genetics 172, 2185-2200
   Abstract »    Full Text »    PDF »

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882