DNA damage and mutagenesis of lambda phage induced by gamma-rays

Abstract

Lambda phage DNA was gamma-irradiated in aqueous solution and the amount of radiation-induced strand breakage [double- and single-strand breaks and alkali-labile sites (dsb, ssb, als)] was determined. Twice as much minor structural damage (ssb and als) per lethal hit was found in this DNA compared with DNA from irradiated phage suspensions. The in vitro irradiated DNA was re-packaged into infectious particles ('pack-phage'). The induction of mutations in the cI or cII cistron was scored using SOS-induced host cells. The in vitro prepared particles were found to have second-order kinetics for mutagenesis induced by gamma-rays indicating that two pre-mutational events were necessary to produce a mutation. In contrast, bacteria-free phage suspensions ('lys-phage') showed single hit kinetics for mutagenesis after irradiation. The increase in the mutation rate in the phage particles was mainly due to minor lesions, i.e. ssb, als and unidentified base damage. In lys-phage, mutagenesis might be enhanced by clustered DNA damage--a configuration which does not exist in pack-phage. The loss of infectivity was analysed in comparison with the extent of structural damage. All lesions investigated here contributed to biological inactivation. Minor lesions were tolerated by lambda phage to a limited extent. Major lesions, such as dsb, contributed most to the loss of infectivity and can be considered as lethal events.