Journal Article

Determination and Analysis of Site-Specific125I Decay-Induced DNA Double-Strand Break End-Group Structures

Datta, Kamal, Weinfeld, Michael, Neumann, Ronald D., Winters, Thomas A.

2007

Overview

End groups contribute to the structural complexity of radiation-induced DNA double-strand breaks (DSBs). As such, end-group structures may affect a cell's ability to repair DSBs. The 3'-end groups of strand breaks caused by$\\gamma radiation$, or oxidative processes, under oxygenated aqueous conditions have been shown to be distributed primarily between 3'-phosphoglycolate and 3'-phosphate, with 5'-phosphate ends in both cases. In this study, end groups of the high-LET-like DSBs caused by$^{125}I decay$were investigated. Site-specific DNA double-strand breaks were produced in plasmid pTC27 in the presence or absence of 2 M DMSO by125I-labeled triplex-forming oligonucleotide targeting. End-group structure was assessed enzymatically as a function of the DSB end to serve as a substrate for ligation and various forms of end labeling. Using this approach, we have demonstrated 3'-hydroxyl (3'-OH) and 3'-phosphate (3'-P) end groups and 5'-ends ($\\geq 42\\%$) terminated by phosphate. A32P postlabeling assay failed to detect 3'-phosphoglycolate in a restriction fragment terminated by the$^{125}I-induced DNA$double-strand break, and this is likely due to restricted oxygen diffusion during irradiation as a frozen aqueous solution. Even so, end-group structure and relative distribution varied as a function of the free radical scavenging capacity of the irradiation buffer.

• Publisher: Radiation Research Society
• Subject: DNA / DNA damage / Free radicals / Gels / Irradiation / Ligation / Oxygen / Plasmids / Radiation damage / Scavenging