DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

Abstract

Although RNA and RNA-binding proteins have been linked to double-strand breaks (DSBs), little is known regarding their roles in the cellular response to DSBs and, if any, in the repair process. Here, we provide direct evidence for the presence of RNA-DNA hybrids at DSBs and suggest that binding of RNA to DNA at DSBs may impact repair efficiency. Our data indicate that the RNA-unwinding protein DEAD box 1 (DDX1) is required for efficient DSB repair and cell survival after ionizing radiation (IR), with depletion of DDX1 resulting in reduced DSB repair by homologous recombination (HR). While DDX1 is not essential for end resection, a key step in homology-directed DSB repair, DDX1 is required for maintenance of the single-stranded DNA once generated by end resection. We show that transcription deregulation has a significant effect on DSB repair by HR in DDX1-depleted cells and that RNA-DNA duplexes are elevated at DSBs in DDX1-depleted cells. Based on our combined data, we propose a role for DDX1 in resolving RNA-DNA structures that accumulate at DSBs located at sites of active transcription. Our findings point to a previously uncharacterized requirement for clearing RNA at DSBs for efficient repair by HR.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00415-16.

ACKNOWLEDGMENTS

This work was supported by operating grants from the Alberta Cancer Foundation and Canadian Breast Cancer Foundation. D.R.G. is a recipient of an Alberta Innovates-Health Solutions graduate studentship.

We thank Maria Jasin, Jeremy Stark, Ranjit Bindra, Stephen Leppla, Bradley Carins, Martin Lavin, Kum Kum Khanna, Gordon Chan, and Ajit Sharma for their kind donations of cell lines and antibodies. We are indebted to Ismail Ismail and Sachin Katyal for helpful comments and sharing unpublished data. We thank Frederic Chedin for sharing the DRIP protocol. We are grateful to Xuejun Sun and Gerry Barron for help with the Imaris software.

None of the authors involved in the preparation of the manuscript have any conflicts of interest to report.

Additional information

Funding

The Alberta Cancer Foundation (grant number 25033) and the Canadian Breast Cancer Foundation provided funding to Roseline Godbout. The Alberta Innovates Health Solutions provided stipend funding to Devon R. Germain.