A Role for DEAD Box 1 at DNA Double-Strand Breaks

Abstract

DEAD box proteins are a family of putative RNA helicases associated with all aspects of cellular metabolism involving the modification of RNA secondary structure. DDX1 is a member of the DEAD box protein family that is overexpressed in a subset of retinoblastoma and neuroblastoma cell lines and tumors. DDX1 is found primarily in the nucleus, where it forms two to four large aggregates called DDX1 bodies. Here, we report a rapid redistribution of DDX1 in cells exposed to ionizing radiation, resulting in the formation of numerous foci that colocalize with γ-H2AX and phosphorylated ATM foci at sites of DNA double-strand breaks (DSBs). The formation of DDX1 ionizing-radiation-induced foci (IRIF) is dependent on ATM, which was shown to phosphorylate DDX1 both in vitro and in vivo. The treatment of cells with RNase H prevented the formation of DDX1 IRIF, suggesting that DDX1 is recruited to sites of DNA damage containing RNA-DNA structures. We have shown that DDX1 has RNase activity toward single-stranded RNA, as well as ADP-dependent RNA-DNA- and RNA-RNA-unwinding activities. We propose that DDX1 plays an RNA clearance role at DSB sites, thereby facilitating the template-guided repair of transcriptionally active regions of the genome.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank Joan Turner and Rufus Day for the M059J and M059K malignant glioma cell lines, Susan Lees-Miller for the BT and L3 lymphoblastoid cell lines, Yosef Shiloh for the EBS and YZ5 cell lines, Razmik Mirzayans for the AT2BE and AT5BI fibroblast cultures from A-T patients, Charlotte Spencer for the anti-RNA polymerase II antibody, James L. Manley for the anti-CstF64 antibody, Roel van Driel for the anti-PML antibody, Joan Steitz for the anti-Sm Y12 antibody, Joan Turner and Jianxun Han for the anti-ATM antibody used for the immunoprecipitation experiments, and Charles Holmes for the microcystin-LR. We are grateful to Kenneth Roy for expert technical assistance. We are indebted to Sachin Katyal, Joan Turner, Michael Hendzed, and Linda Reha-Krantz for helpful comments.

This work was supported by a grant from the Alberta Cancer Research Institute. L.L. is the recipient of an Alberta Cancer Research Institute fellowship.