The human ATR-mediated DNA damage checkpoint in a reconstituted system
Abstract
DNA damage checkpoints are signal transduction pathways that coordinate the cell cycle with other cellular responses to DNA damage in order to maintain genomic integrity. The ATR kinase is responsible for initiating the DNA damage checkpoint in response to UV-damaged DNA and incompletely replicated DNA. We have recently established an in vitro system that recapitulates most but not all features of the human ATR-mediated DNA damage checkpoint response. With this system we have shown that damaged DNA can be a direct signal for ATR activation, and that TopBP1 specifically stimulates ATR in the presence of damaged DNA under physiological ionic strength conditions. This system provides a powerful tool to gain insight into the molecular mechanism of the ATR pathway. Here we describe preparation of the checkpoint components and our specific kinase assay in more detail.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
ATR in complex with ATRIP binds RPA-coated single-stranded DNA through an ATRIP-RPA interaction, and ATRIP activates ATR checkpoint kinase activity.
“ATR in complex with its nuclear binding partner ATR-interacting protein (ATRIP) binds to this RPA-coated ssDNA via an ATRIP-RPA interaction. ATRIP also serves to activate the checkpoint kinase activity of ATR 4, 27–30.”
From Xeroderma Pigmentosa Group A (XPA), Nucleotide Excision Repair and Regulation by ATR in Response to Ultraviolet Irradiation (2017) · cites this paper as [28]
Cites
Molecular Mechanisms of Mammalian DNA Repair and the DNA Damage Checkpoints
Sancar, A., Lindsey-Boltz, L. A., Ünsal-Kaçmaz, K., & Linn, S. (2004) · Annual Review of Biochemistry · doi:10.1146/annurev.biochem.73.011303.073723 · Cited by 3,317 · see citing works
Self-citationCell cycle checkpoint signaling through the ATM and ATR kinases
Abraham, R. T. (2001) · Genes & Development · doi:10.1101/gad.914401 · Cited by 1,966 · see citing works
ATR: an essential regulator of genome integrity
Cimprich, K. A., & Cortez, D. (2008) · Nature Reviews Molecular Cell Biology · doi:10.1038/nrm2450 · Cited by 1,798 · see citing works
Liu, Q., Guntuku, S., Cui, X.-S., Matsuoka, S., Cortez, D., Tamai, K., Luo, G., Carattini-Rivera, S., DeMayo, F., Bradley, A., et al. (2000) · Genes & Development · doi:10.1101/gad.14.12.1448 · Cited by 1,625 · see citing works
Metabolic activation of benzo(a)pyrene proceeds by a diol-epoxide
Sims, P. H., Grover, P. L., Swaisland, A. J., Pal, K., & Hewer, A. J. (1974) · Nature · doi:10.1038/252326a0 · Cited by 1,134 · see citing works
ATR-Mediated Checkpoint Pathways Regulate Phosphorylation and Activation of Human Chk1
Zhao, H., & Piwnica-Worms, H. (2001) · Molecular and Cellular Biology · doi:10.1128/mcb.21.13.4129-4139.2001 · Cited by 1,091 · see citing works
ATR and ATRIP: Partners in Checkpoint Signaling
Cortez, D., Guntuku, S., Qin, J., & Elledge, S. J. (2001) · Science · doi:10.1126/science.1065521 · Cited by 985 · see citing works
TopBP1 Activates the ATR-ATRIP Complex
Kumagai, A., Lee, J., Yoo, H. Y., & Dunphy, W. G. (2006) · Cell · doi:10.1016/j.cell.2005.12.041 · Cited by 743 · see citing works
Essential role of BETA2/NeuroD1 in development of the vestibular and auditory systemsPreprint
Liu, M., Pereira, F. A., Price, S. D., Chu, M.-J., Shope, C. D., Himes, D., Eatock, R. A., Brownell, W. E., Lysakowski, A., & Tsai, M. (2000) · Genes & Development · doi:10.1101/gad.840500 · Cited by 625 · see citing works
Benzo[ a ]pyrene Diol Epoxides as Intermediates in Nucleic Acid Binding in Vitro and in Vivo
Weinstein, I. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Harvey, R. G., Harris, C. C., Autrup, H. N., Kasai, H., & Nakanishi, K. (1976) · Science · doi:10.1126/science.959820 · Cited by 578 · see citing works
Guo, Z., Kumagai, A., Wang, S. X., & Dunphy, W. G. (2000) · Genes & Development · doi:10.1101/gad.842500 · Cited by 417 · see citing works
TopBP1 activates ATR through ATRIP and a PIKK regulatory domainPreprint
Mordes, D. A., Glick, G. G., Zhao, R., & Cortez, D. (2008) · Genes & Development · doi:10.1101/gad.1666208 · Cited by 368 · see citing works
Kriek, E., MILLER, J. A., Juhl, U., & Miller, E. C. (1967) · Biochemistry · doi:10.1021/bi00853a029 · Cited by 329 · see citing works
[35] In Vitro transcription: Whole-cell extract
Manley, J. L., Fire, A. Z., Samuels, M. E., & Sharp, P. A. (1983) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/0076-6879(83)01038-1 · Cited by 294 · see citing works
DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[a]pyrenedihydrodiol epoxide
Cheng, S. C., Hilton, B. D., Roman, J. M., & Dipple, A. (1989) · Chemical Research in Toxicology · doi:10.1021/tx00011a011 · Cited by 279 · see citing works
Slaga, T. J., Bracken, W. J., Gleason, G. L., Levin, W., Yagi, H., JERINA, D. M., & Conney, A. H. (1979) · PubMed · Cited by 247 · see citing works
Double-stranded DNA stereoselectively binds benzo(a)pyrene diol epoxides
Meehan, T. D., & Straub, K. M. (1979) · Nature · doi:10.1038/277410a0 · Cited by 215 · see citing works
Recognition of DNA Adducts by Human Nucleotide Excision Repair
Gunz, D., Heß, M., & Naegeli, H. (1996) · Journal of Biological Chemistry · doi:10.1074/jbc.271.41.25089 · Cited by 197 · see citing works
Preferential binding of ATR protein to UV-damaged DNA
Ünsal-Kaçmaz, K., Makhov, A. M., Griffith, J. D., & Sancar, A. (2002) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.102167799 · Cited by 176 · see citing works
Self-citationIdentification and functional analysis of TopBP1 and its homologs
Garcia, V., Furuya, K., & Carr, A. M. (2005) · DNA Repair · doi:10.1016/j.dnarep.2005.04.001 · Cited by 158 · see citing works
Carcinogenicity of benzo[a]pyrene 4,5-, 7,8-, and 9,10-oxides on mouse skin.
Levin, W., Wood, A. W., Yagi, H., Dansette, P. M., JERINA, D. M., & Conney, A. H. (1976) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.73.1.243 · Cited by 123 · see citing works
Dpb11 activates the Mec1-Ddc2 complex
Mordes, D. A., Nam, E. A., & Cortez, D. (2008) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0806621105 · Cited by 121 · see citing works
Yeast DNA replication protein Dpb11 activates the Mec1/ATR checkpoint kinase
Navadgi-Patil, V. M., & Burgers, P. M. (2008) · Journal of Biological Chemistry · doi:10.1074/jbc.m807435200 · Cited by 121 · see citing works
Yamane, K., Kawabata, M., & TSURUO, T. (1997) · European Journal of Biochemistry · doi:10.1111/j.1432-1033.1997.00794.x · Cited by 119 · see citing works
Ünsal-Kaçmaz, K., & Sancar, A. (2004) · Molecular and Cellular Biology · doi:10.1128/mcb.24.3.1292-1300.2003 · Cited by 103 · see citing works
Self-citationRecruitment of DNA damage checkpoint proteins to damage in transcribed and nontranscribed sequences
Jiang, G., & Sancar, A. (2006) · Molecular and Cellular Biology · doi:10.1128/mcb.26.1.39-49.2006 · Cited by 70 · see citing works
Self-citationReconstitution of a human ATR-mediated checkpoint response to damaged DNA.
Choi, J.-H., Lindsey-Boltz, L. A., & Sancar, A. (2007) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0706013104 · Cited by 64 · see citing works
Self-citationHashimoto, Y., Tsujimura, T., Sugino, A., & Takisawa, H. (2006) · Genes to Cells · doi:10.1111/j.1365-2443.2006.00998.x · Cited by 63 · see citing works
Cooperative activation of the ATR checkpoint kinase by TopBP1 and damaged DNA
Choi, J.-H., Lindsey‐Boltz, L. A., & Sancar, A. (2009) · Nucleic Acids Research · doi:10.1093/nar/gkn1075 · Cited by 46 · see citing works
Self-citationA Repair Competition Assay to Assess Recognition by Human Nucleotide Excision Repair
Hess, M. T., Gunz, D., & Naegeli, H. (1996) · Nucleic Acids Research · doi:10.1093/nar/24.5.824 · Cited by 22 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling
Choi, J.-H., Lindsey‐Boltz, L. A., Kemp, M. G., Mason, A. C., Wold, M. S., & Sancar, A. (2010) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1007856107 · Cited by 144 · see citing works
“The kinase activity of the ATR-containing fractions was analyzed using Chk1 as a substrate as described previously (23, 32, 39), with”
MethodSelf-citationChen, Y., Chiang, S., Lin, J., Yang, J., Ma, Y., Liao, C., Lai, T.-Y., Tang, N.-Y., & Chung, J. (2010) · PubMed · Cited by 91 · see citing works
Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System
Lindsey-Boltz, L. A., Kemp, M. G., Reardon, J. T., DeRocco, V., Iyer, R. R., Modrich, P., & Sancar, A. (2014) · Journal of Biological Chemistry · doi:10.1074/jbc.m113.542787 · Cited by 59 · see citing works
Self-citationLindsey‐Boltz, L. A., Serçin, Ö., Choi, J.-H., & Sancar, A. (2009) · Journal of Biological Chemistry · doi:10.1074/jbc.m109.064485 · Cited by 55 · see citing works
Self-citationThe human intra-S checkpoint response to UVC-induced DNA damage
Kaufmann, W. K. (2009) · Carcinogenesis · doi:10.1093/carcin/bgp230 · Cited by 52 · see citing works
Wu, L., Lu, H.-F., Chou, Y., Shih, Y., Bau, D., Chen, J., Hsu, S., & Chung, J. (2015) · The American Journal of Chinese Medicine · doi:10.1142/s0192415x1550024x · Cited by 42 · see citing works
JI, B.-C., Yu, C.-C., Yang, S., Hsia, T., Yang, J., Lai, K., Ko, Y., Lin, J., Lai, T.-Y., & Chung, J. (2012) · Oncology Reports · doi:10.3892/or.2012.1622 · Cited by 40 · see citing works
Lu, H.-F., Lai, T. Y., Hsia, T.-C., Tang, Y.-J., Yang, J., Chiang, J., Lu, C., Liu, C. M., Wang, H.-L., & Chung, J. (2010) · Neurochemical Research · doi:10.1007/s11064-010-0161-z · Cited by 38 · see citing works
“point for UV-damaged DNA and incompletely replicated DNA [22].”
BackgroundLindsey‐Boltz, L. A., & Sancar, A. (2011) · Journal of Biological Chemistry · doi:10.1074/jbc.m111.237958 · Cited by 35 · see citing works
Self-citationHsia, T., Lin, J.-H., Hsu, S., Tang, N.-Y., Lu, H.-F., Wu, S., Lin, J., & Chung, J. (2014) · Environmental Toxicology · doi:10.1002/tox.21986 · Cited by 35 · see citing works
“ATM and ATR, that are two master checkpoint kinases activated by double-stranded DNA breaks (DSBs) (Choi et al., 2009; Shiotani and Zou, 2009).”
BackgroundLindsey‐Boltz, L. A., Reardon, J. T., Wold, M. S., & Sancar, A. (2012) · Journal of Biological Chemistry · doi:10.1074/jbc.m112.407825 · Cited by 30 · see citing works
Self-citationMusich, P. R., Li, Z., & Zou, Y. (2017) · Advances in Experimental Medicine and Biology · doi:10.1007/978-3-319-56017-5_4 · Cited by 23 · see citing works
Propofol induces DNA damage in mouse leukemic monocyte macrophage RAW264.7 cells
Wu, K., Yang, S., Hsu, S., Chiang, J., Hsia, T., Yang, J., Liu, K.-C., Wu, R. S.-C., & Chung, J. (2013) · Oncology Reports · doi:10.3892/or.2013.2722 · Cited by 23 · see citing works
Lin, J., Wu, C., Hsu, S., Weng, S., Ma, Y., Huang, Y., Lin, J., & Chung, J. (2014) · Environmental Toxicology · doi:10.1002/tox.22003 · Cited by 19 · see citing works
“This is in agreement with other reports which showed that agents induced doublestranded DNA breaks which can activate two master checkpoint kinases (ATM and ATR) (Choi et al., 2009; Shiotani and Zou, 2009) for cells keeping genomic integrity.”
ResultInfluentialA new in vitro system for activating the cell cycle checkpoint
Wang, J., Engle, S. E., & Zhang, Y. (2011) · Cell Cycle · doi:10.4161/cc.10.3.14753 · Cited by 17 · see citing works
“In contrast, such protein phosphorylation can occur without damaged DNA when purified proteins were used, although addition of damaged DNA is able to enhance the phosphorylation extent.(38,39) In this regard, the IVTNT system is a combination of these two assays, i.”
Hassan, B. H., Lindsey‐Boltz, L. A., Kemp, M. G., & Sancar, A. (2013) · Journal of Biological Chemistry · doi:10.1074/jbc.m113.475517 · Cited by 17 · see citing works
Self-citationWD40-repeat protein WDR18 collaborates with TopBP1 to facilitate DNA damage checkpoint signaling
Yan, S., & Willis, J. (2013) · Biochemical and Biophysical Research Communications · doi:10.1016/j.bbrc.2012.12.144 · Cited by 16 · see citing works
Multiple ATR-Chk1 Pathway Proteins Preferentially Associate with Checkpoint-Inducing DNA Substrates
Yilmaz, S., Sancar, A., & Kemp, M. G. (2011) · PLoS ONE · doi:10.1371/journal.pone.0022986 · Cited by 12 · see citing works
“kinase stimulation by bulky adduct-containing DNA [9,10,29]”
BackgroundSelf-citationForma, E., Bryś, M., & Wanda, M. (2011) · InTech eBooks · doi:10.5772/22274 · Cited by 11 · see citing works
“ATM and ATR are proteins of about 300 kDa, with a conserved C-terminal catalytic domain that preferably phosphorylates serine or threonine residues followed by a glutamine, i.e. SQ or TQ motif (Choi et al., 2009; Smits et al., 2010).”
BackgroundBringing It All Together: Coupling Excision Repair to the DNA Damage Checkpoint
Lindsey‐Boltz, L. A. (2016) · Photochemistry and Photobiology · doi:10.1111/php.12667 · Cited by 11 · see citing works
Self-citationJordan, M. R., Oakley, G. G., Mayo, L. D., Balakrishnan, L., & Turchi, J. J. (2024) · Scientific Reports · doi:10.1038/s41598-024-70589-y · Cited by 7 · see citing works
Zhang, Y., & Gong, F. (2009) · Methods · doi:10.1016/j.ymeth.2009.05.001 · Cited by 2 · see citing works
Synthesis and biological evaluation of novel anti-tumour (E)-styrylsulfonyl methylpyridines
Lu, T. (2014) · Nottingham ePrints (University of Nottingham)
- Authors
- Choi, J.-H., Sancar, A., & Lindsey-Boltz, L. A.
- Affiliations
- University of North Carolina at Chapel Hill (US)
- ORCID
- Sancar, Aziz
- Venue
- Methods
- Publisher
- Elsevier BV
- Published
- 2009-05
- Volume
- 48
- Issue
- 1
- Pages
- 3-7
- ISSN
- 1046-2023 (ISSN-L)
- OpenAlex
- W2058564277
- Cited by
- 23
- References
- 30
- Citations by year
- 2024: 1 · 2017: 1 · 2016: 1 · 2015: 1 · 2014: 4 · 2013: 3 · 2012: 2
- FWCI
- 1.61
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Methods (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of General Medical Sciences (R01GM032833)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Keywords
- ATR kinase, DNA damage checkpoint, TopBP1, UV-induced DNA damage, in vitro reconstitution, DNA damage signaling, kinase activity assay, cell cycle checkpoints, genome stability
- MeSH terms
- Checkpoint Kinase 1, Carrier Proteins, Cell-Free System, DNA, DNA Damage, DNA-Binding Proteins, Glutathione Transferase, HeLa Cells, Histidine, Humans, Models, Biological, Molecular Weight, Nuclear Proteins, Osmolar Concentration, Phosphorylation, Protein Binding, Protein Kinases, Recombinant Proteins, Substrate Specificity, Silver Staining, Protein Serine-Threonine Kinases, Protein Structure, Tertiary, Cell Cycle Proteins, Amino Acid Motifs, Adaptor Proteins, Signal Transducing, Ataxia Telangiectasia Mutated Proteins
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
