In vitro mutagenesis as a result of 60Co-gamma-ray-induced base damage

We utilized a model system to study the mechanism(s) of mutation resulting from gamma-ray-induced DNA base damage. 60Co-irradiated, uracil-containing M13mp2 DNA was hybridized to normal (non-uracil) linearized double-stranded virus DNA minus the lac reporter region. Only DNA without strand breaks in the reporter region will circularize. This DNA was used as a substrate for a modified T7 DNA polymerase with no residual 3'----5' exonuclease activity (Sequenase 2). The reaction product was transfected into a rec- bacterial host to minimize the occurrence of bypass events in vivo, and mutant progeny were selected. DNA irradiated with 400 or 800 Gy from a 60Co gamma-source gave about a 5-fold increase in the percentage of mutants recovered after synthesis with Sequenase as compared to the recovery of mutants using control DNA. About 20% of the mutants recovered from both irradiated and control templates contained multiple mutations in the target area sequenced. The irradiated samples had an excess of mutations which resulted from changes at pyrimidines. C---T transitions were most common. Mutations at T were mostly (-1) and (-2) frameshifts, particularly at sequences of repeated T's.