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

Thomas F. Martínez, John W. Phillips, Kenneth K. Karanja, Piotr Polaczek, Chieh Mei Wang, Benjamin C. Li, Judith L. Campbell, Peter B. Dervan

Research output: Contribution to journalArticle

17 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)11546-11559
Number of pages14
JournalNucleic acids research
Volume42
Issue number18
DOIs
StatePublished - Oct 13 2014

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