Determination and analysis of site-specific 125I decay-induced DNA double-strand break end-group structures.
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 125I decay were investigated. Site-specific DNA double-strand breaks were produced in plasmid pTC27 in the presence or absence of 2 M DMSO by 125I-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 (> or = 42%) terminated by phosphate. A 32P postlabeling assay failed to detect 3'-phosphoglycolate in a restriction fragment terminated by the 125I-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.
Datta
Kamal
K
Nuclear Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.
Weinfeld
Michael
M
Neumann
Ronald D
RD
Winters
Thomas A
TA
D001483:Base Sequence; D004249:DNA Damage; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005609:Free Radicals; D007457:Iodine Radioisotopes / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D013379:Substrate Specificity
D005609:Free Radicals; D007457:Iodine Radioisotopes; D009841:Oligonucleotides; D010710:Phosphates; D004262:DNA Restriction Enzymes; D011088:DNA Ligases; D010100:Oxygen; D004121:Dimethyl Sulfoxide