title: Replication stress by Py-Im polyamides induces a non-canonical ATR-dependent checkpoint response.
abstract: 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.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Martínez
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Phillips
authors.affiliation: Braun Laboratories, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Karanja
authors.affiliation: Braun Laboratories, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Piotr
authors.initials: P
authors.lastname: Polaczek
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Chieh-Mei
authors.initials: CM
authors.lastname: Wang
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Li
authors.affiliation: Braun Laboratories, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Judith L
authors.initials: JL
authors.lastname: Campbell
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA dervan@caltech.edu.
authors.forename: Peter B
authors.initials: PB
authors.lastname: Dervan
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D002460:Cell Line; D064447:Checkpoint Kinase 2 / Q000378:metabolism; D053960:DNA Breaks; D004265:DNA Helicases / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication / Q000187:drug effects*; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000633:toxicity*; D064111:Minichromosome Maintenance Complex Component 2 / Q000378:metabolism; D009757:Nylons / Q000633:toxicity*; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D011758:Pyrroles / Q000633:toxicity*; D051716:Replication Protein A / Q000378:metabolism; D059807:S Phase Cell Cycle Checkpoints / Q000187:drug effects*; D013312:Stress, Physiological / Q000235:genetics; D054875:Ubiquitination
chemical_list: D052236:Fanconi Anemia Complementation Group D2 Protein; D007093:Imidazoles; D009757:Nylons; D018809:Proliferating Cell Nuclear Antigen; D011758:Pyrroles; D051716:Replication Protein A; D064447:Checkpoint Kinase 2; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D004265:DNA Helicases; D064111:Minichromosome Maintenance Complex Component 2