DNA double-strand breaks are considered to be the most deleterious lesion 
induced by ionizing radiation. However, the mechanism of rejoining of these 
lesions has not been extensively studied at the molecular level. We have used a 
shuttle vector, pHAZE, to analyze the mechanism of rejoining of DNA 
double-strand breaks in human cells. The advantage of this vector system is 
that, unlike many previously described shuttle vectors, it has a large target 
gene for the detection of deletions and it is maintained as a freely replicating 
episome with chromatin conformation in the nucleus of human cells. In this study 
we compare data obtained on the spectrum of mutations induced in pHAZE by 
ionizing radiation (alpha-particles) and restriction enzymes (PvuII, ClaI, and 
PvuI). Unlike ionizing radiation, restriction enzymes induce double-strand 
breaks in DNA with known end structures at defined locations and therefore 
provide a model system for analyzing cellular responses to DNA double-strand 
breaks. Exposure of human cells containing the vector to alpha-particle 
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 junction region. Analysis of 
restriction-enzyme-induced mutations suggests 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 fewer modifications introduced at the break points before 
rejoining than breaks with blunt ends. When considered in relation to the data 
obtained with ionizing radiation this suggests that the presence of cohesive 
sequences either at, or in proximity to, the ends enhances rejoining of DNA 
double-strand breaks.