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Updated: Jul 2, 2026
Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins
Published on: September 28, 2012
A role for DEAD box 1 at DNA double-strand breaks
Lei Li1, Elizabeth A Monckton, Roseline Godbout
1Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.
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 gamma-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.
Insights
DEAD box protein DDX1 rapidly forms foci at DNA double-strand breaks (DSBs) after radiation. This ATM-dependent process involves RNA-DNA structures, suggesting DDX1 clears RNA to aid DNA repair.
Area of Science:
- Molecular Biology
- Cellular Metabolism
- DNA Damage Response
Background:
- DEAD box proteins are RNA helicases crucial for RNA metabolism.
- DDX1, a DEAD box protein, is overexpressed in certain cancers and forms nuclear aggregates (DDX1 bodies).
Purpose of the Study:
- To investigate the role and localization of DDX1 in response to DNA damage.
- To elucidate the mechanism of DDX1 recruitment to sites of DNA double-strand breaks (DSBs).
Main Methods:
- Immunofluorescence microscopy to observe DDX1 foci formation and colocalization with DNA damage markers (gamma-H2AX, p-ATM).
- In vitro and in vivo kinase assays to assess ATM-dependent phosphorylation of DDX1.
- RNase H treatment to evaluate the role of RNA-DNA structures in DDX1 recruitment.
Main Results:
- Ionizing radiation induces rapid redistribution of DDX1 into foci colocalizing with gamma-H2AX and p-ATM at DSB sites.
- DDX1 foci formation (IRIF) is dependent on ATM kinase activity and DDX1 phosphorylation.
- RNase H treatment inhibits DDX1 IRIF formation, indicating recruitment to RNA-containing DNA damage sites.
- DDX1 exhibits RNase activity and unwinding activity on RNA-DNA and RNA-RNA hybrids.
Conclusions:
- DDX1 is recruited to DSBs in an ATM-dependent manner, involving RNA-DNA structures.
- DDX1 possesses RNA helicase and RNase activities, suggesting a role in RNA processing at DNA damage sites.
- DDX1 may function in RNA clearance at DSBs to facilitate efficient DNA repair, particularly in transcriptionally active genomic regions.
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