MECHANISMS INVOLVED IN REJOINING DNA DOUBLE-STRAND BREAKS INDUCED BY IONIZING-RADIATION AND RESTRICTION ENZYMES

DNA double-strand breaks are considered to be the most deleterious les ion induced by ionizing radiation. However, the mechanism of rejoining of these lesions has not been extensively studied at the molecular le vel. We have used a shuttle vector, pHAZE, to analyze the mechanism of rejoining of DNA double-strand breaks in human cells. The advantage o f this vector system is that, unlike many previously described shuttle vectors, it has a large target gene for the detection of deletions an d it is maintained as a freely replicating episome with chromatin conf ormation in the nucleus of human cells. In this study we compare data obtained on the spectrum of mutations induced in pHAZE by ionizing rad iation (a-particles) and restriction enzymes (PvuII, ClaI, and PvuI). Unlike ionizing radiation, restriction enzymes induce double-strand br eaks in DNA with known end structures at defined locations and therefo re provide a model system for analyzing cellular responses to DNA doub le-strand breaks. Exposure of human cells containing the vector to a-p article irradiation produced both point mutations and large deletions in pHAZE. When the junction regions of the deletions were sequenced it was found that 65% were rejoined with up to 6 bp of homology at the j unction region. Analysis of restriction-enzyme-induced mutations sugge sts that double-strand break ends are modified to facilitate rejoining and that the type of modification is characteristic for different end structures. Double-strand breaks with cohesive ends appear to have fe wer modifications introduced at the break points before rejoining than breaks with blunt ends. When considered in relation to the data obtai ned with ionizing radiation this suggests that the presence of cohesiv e sequences either at, or in proximity to, the ends enhances rejoining of DNA double-strand breaks.