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Replication stress by Py–Im polyamides induces a non-canonical ATR-dependent checkpoint response

Martínez, Thomas F. and Philips, John W. and Karanja, Kenneth K. and Polaczek, Piotr and Wang, Chieh-Mei and Li, Benjamin C. and Campbell, Judith L. and Dervan, Peter B. (2014) Replication stress by Py–Im polyamides induces a non-canonical ATR-dependent checkpoint response. Nucleic Acids Research, 42 (18). pp. 11546-11559. ISSN 0305-1048. PMCID PMC4191428.

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Pyrrole–imidazole polyamides targeted to the androgen response element were cytotoxic in multiple cell lines, independent of intact androgen receptor signaling. Polyamide treatment induced accumulation of S-phase cells and of PCNA replication/repair foci. Activation of a cell cycle checkpoint response was evidenced by autophosphorylation of ATR, the S-phase checkpoint kinase, and by recruitment of ATR and the ATR activators RPA, 9-1-1, and Rad17 to chromatin. Surprisingly, ATR activation was accompanied by only a slight increase in single-stranded DNA, and the ATR targets RPA2 and Chk1, a cell cycle checkpoint kinase, were not phosphorylated. However, ATR activation resulted in phosphorylation of the replicative helicase subunit MCM2, an ATR effector. Polyamide treatment also induced accumulation of monoubiquitinated FANCD2, which is recruited to stalled replication forks and interacts transiently with phospho-MCM2. This suggests that polyamides induce replication stress that ATR can counteract independently of Chk1 and that the FA/BRCA pathway may also be involved in the response to polyamides. In biochemical assays, polyamides inhibit DNA helicases, providing a plausible mechanism for S-phase inhibition.

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Dervan, Peter B.0000-0001-8852-7306
Additional Information:© 2014 The Author(s). Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Received April 22, 2014; Revised September 09, 2014; Accepted September 10, 2014. First published online: September 23, 2014. We thank Dr. Subhash Pokharel for S. cerevisiae Dna2-K677R, C.L. Sawyers (University of California, Los Angeles) for the LNAR cell line and L. Zou for the ATRpT1989 antibody (Harvard Medical School). We thank members of the Dervan and Campbell laboratories for critical reading of the manuscript. We thank members of the Dunphy laboratory for helpful discussions. Mass spectrometry analyses were performed in the Mass Spectrometry Laboratory of the Division of Chemistry and Chemical Engineering at the California Institute of Technology. Imaging was performed at the Caltech Bioimaging Center in the Beckman Institute. Flow cytometry analyses were performed at the Caltech Flow Cytometry Cell Sorting Facility. We thank Rochelle Diamond for support and technical expertise in performing flow cytometry experiments. FUNDING: National Institutes of Health (NIH) [R01GM27681, R01GM51747 to P.B.D.; R01GM07866 to J.L.C.]; Ellison Foundation [AG-SS-2143-08 to J.L.C.]; Margaret Early Trust [to J.L.C.]; Army Research Office [09-1-0041 to J.L.C.]; NIH [F31CA159896 to T.F.M.]. Funding for open access charge: NIH [R01GM027681 to P.B.D.]. Conflict of interest statement. None declared.
Funding AgencyGrant Number
Ellison FoundationAG-SS-2143-08
Margaret Early TrustUNSPECIFIED
Army Research Office (ARO)09-1-0041
Issue or Number:18
PubMed Central ID:PMC4191428
Record Number:CaltechAUTHORS:20140929-091558705
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Official Citation:Thomas F. Martínez, John W. Phillips, Kenneth K. Karanja, Piotr Polaczek, Chieh-Mei Wang, Benjamin C. Li, Judith L. Campbell, and Peter B. Dervan Replication stress by Py–Im polyamides induces a non-canonical ATR-dependent checkpoint response Nucl. Acids Res. (13 October 2014) 42 (18): 11546-11559 first published online September 23, 2014 doi:10.1093/nar/gku866
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:50086
Deposited By: Jason Perez
Deposited On:29 Sep 2014 18:12
Last Modified:26 Nov 2019 11:15

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