The clastogenicity of two
restriction endonucleases with almost equal cutting frequencies: PvuII, generating blunt-ended
DNA double-strand breaks (
dsb) and EcoRI, generating cohesive-ended
dsb, has been measured
after treatment of electroporated CHO cells with these
enzymes. Chromosome damage was assessed by the micronucleus cytokinesis-block technique, and for certain electroporation voltages by analysis of metaphase preparations. As has been found in previous studies, PvuII was found to be more effective in causing chromosomal damage than EcoRI, indicating the greater importance of blunt-ended
dsb in
chromosome aberration induction. These findings also validate the use of the micronucleus cytokinesis-block technique for evaluating chromosomal damage from
restriction endonucleases. The results show a biphasic induction of micronuclei in binucleate cells as a function of time
after treatment. This pattern is interpreted as indicating variable sensitivity of cells to
restriction endonucleases at different stages of the cell cycle. The micronucleus data show that late collection times (40-48 h
after treatment) give higher frequencies than short times. Both micronucleus and metaphase aberration data indicate that voltages in excess of 260 V are more efficient in porating cells than lower voltages and, as a result, lower
restriction endonuclease concentrations could be used.