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Small-molecule–induced DNA damage identifies alternative DNA structures in human genes

Nature Chemical Biology volume 8, pages 301–310 (2012)Cite this article

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Abstract

Guanine-rich DNA sequences that can adopt non–Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here we show that the G-quadruplex–interacting drug pyridostatin promotes growth arrest in human cancer cells by inducing replication- and transcription-dependent DNA damage. A chromatin immunoprecipitation sequencing analysis of the DNA damage marker γH2AX provided the genome-wide distribution of pyridostatin-induced sites of damage and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein abundance and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target of this drug. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions.

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Figure 1: Pyridostatin-induced DNA damage and checkpoint-dependent cell-cycle arrest.
Figure 2: Pyridostatin-induced transcription- and replication-dependent DNA damage.
Figure 3: Visual analysis of pyridostatin targets.
Figure 4: Pyridostatin and hPif1 targeted overlapping sites in cells.
Figure 5: ChIP-seq analysis identified genomic targets of pyridostatin.
Figure 6: Pyridostatin interacted with G-quadruplex motifs in SRC.
Figure 7: Pyridostatin targeted the proto-oncogene SRC.

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Acknowledgements

We thank AstraZeneca for providing the Chk1/ Chk2i (AZD7762), S. Müller for technical assistance with FRET melting experiments and the US National Cancer Institute for screening pyridostatin on a panel of 60 cancer-cell lines. R.R. is a Herchel Smith Research Fellow, K.M.M. is funded by the Wellcome Trust project grant 086861/Z/08/Z, and J.V.F. is funded by Cancer Research UK (CRUK) program grant C6/A11224. Research in the S. Balasubramanian laboratory is funded by CRUK program grant C9681/A11961 and a project grant from the Biotechnology and Biological Sciences Research Council (BB/G008337/1). S. Britton is funded by an EMBO long-term fellowship (ALTF 93-2010). Research in the S.P.J. laboratory is funded by CRUK program grant C6/A11224, the European Research Council and the European Community Seventh Framework Programme (GENICA and DDResponse). Core infrastructure funding was provided by CRUK and the Wellcome Trust. S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK.

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Author notes

  1. Raphaël Rodriguez and Kyle M Miller: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, University of Cambridge, Cambridge, UK

    Raphaël Rodriguez, Mehran Nikan & Shankar Balasubramanian

  2. The Gurdon Institute, University of Cambridge, Cambridge, UK

    Raphaël Rodriguez, Kyle M Miller, Josep V Forment, Charles R Bradshaw, Sébastien Britton, Tobias Oelschlaegel, Blerta Xhemalce & Stephen P Jackson

  3. Department of Biochemistry, University of Cambridge, Cambridge, UK

    Kyle M Miller, Josep V Forment, Sébastien Britton, Tobias Oelschlaegel & Stephen P Jackson

  4. Department of Molecular Genetics and Microbiology, Institute for Molecular and Cellular Biology, University of Texas at Austin, Austin, Texas, USA

    Kyle M Miller & Blerta Xhemalce

  5. Cancer Research UK, Cambridge Research Institute, Li Ka Shing Center, Cambridge, UK

    Shankar Balasubramanian

  6. School of Clinical Medicine, University of Cambridge, Cambridge, UK

    Shankar Balasubramanian

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Contributions

R.R., K.M.M., S. Balasubramanian and S.P.J. conceptualized the study. R.R., K.M.M. and S.P.J. designed the experiments. R.R. and K.M.M. performed all the experiments, unless started otherwise. J.V.F. provided assistance with western blotting and the fluorescence-activated cell sorting analyses. R.R. invented pyridostatin and pyridostatin-β and developed the cellular labeling strategy. M.N. performed the chemical synthesis of pyridostatin-β. T.O. and S. Britton performed the hPif1 experiments. R.R. and M.N. performed the circular dichroism and NMR spectroscopy. C.R.B. performed the computational data analysis. B.X. provided assistance with ChIP-seq, qRT-PCR and the wound healing assays. All the authors analyzed the data. K.M.M., R.R. and S.P.J. wrote the manuscript.

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Correspondence to Shankar Balasubramanian or Stephen P Jackson.

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Rodriguez, R., Miller, K., Forment, J. et al. Small-molecule–induced DNA damage identifies alternative DNA structures in human genes. Nat Chem Biol 8, 301–310 (2012). https://doi.org/10.1038/nchembio.780

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