Repair of radiation-induced DNA double-strand breaks is dependent upon radiation quality and the structural complexity of double-strand breaks.
Mammalian cells primarily repair DSBs by nonhomologous end joining (NHEJ). To assess the ability of human cells to mediate end joining of complex DSBs such as those produced by chemicals, oxidative events, or high- and low-LET radiation, we employed an in vitro double-strand break repair assay using plasmid DNA linearized by these various agents. We found that human HeLa cell extracts support end joining of complex DSBs and form multimeric plasmid products from substrates produced by the radiomimetic drug bleomycin, 60Co gamma rays, and the effects of 125I decay in DNA. End joining was found to be dependent on the type of DSB-damaging agent, and it decreased as the cytotoxicity of the DSB-inducing agent increased. In addition to the inhibitory effects of DSB end-group structures on repair, NHEJ was found to be strongly inhibited by lesions proximal to DSB ends. The initial repair rate for complex non-ligatable bleomycin-induced DSBs was sixfold less than that of similarly configured (blunt-ended) but less complex (ligatable) restriction enzyme-induced DSBs. Repair of DSBs produced by gamma rays was 15-fold less efficient than repair of restriction enzyme-induced DSBs. Repair of the DSBs produced by 125I was near the lower limit of detection in our assay and was at least twofold lower than that of gamma-ray-induced DSBs. In addition, DSB ends produced by 125I were shown to be blocked by 3'-nucleotide fragments: the removal of these by E. coli endonuclease IV permitted ligation.
Pastwa
Elzbieta
E
Nuclear Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.
Neumann
Ronald D
RD
Mezhevaya
Katherina
K
Winters
Thomas A
TA
D001761:Bleomycin / Q000494:pharmacology; D003037:Cobalt Radioisotopes; D004247:DNA / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects*; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000378:metabolism; D005720:Gamma Rays; D006367:HeLa Cells; D006801:Humans; D007457:Iodine Radioisotopes; D008968:Molecular Conformation; D011995:Recombination, Genetic; D013997:Time Factors
D003037:Cobalt Radioisotopes; D007457:Iodine Radioisotopes; D001761:Bleomycin; D004247:DNA; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); D011088:DNA Ligases