DNA repair and mutagenesis of bacteriophage T7

A DNA replication-packaging system of bacteriophage T7 has been developed as a vehicle for studying mutagenesis. With this system, the complete life-cycle of T7 can be performed in vitro and the biological consequences of in vitro manipulations determined by examining viable phage particles. The close resemblance between this system and the in vivo condition was demonstrated in part by in vivo and in vitro host cell reactivation studies of T7 damaged by alkylating agents. Moreover, these studies provided good evidence for in vitro repair of at least some of the lethal damage inflicted by these chemicals and demonstrated significant levels of in vitro DNA synthesis even when alkylated DNA was provided as template. The actual demonstration of mutagenesis was accomplished by including 0⁶methyldeoxyguanosine triphosphate as a precursor during the DNA synthesis reaction and measuring either reversion of an amber mutation or generation of temperature-sensitive mutants in phage produced by in vitro packaging. Until now, only indirect evidence supported the proposal that alkylating agents are mutagenic because of their ability to react at the oxygen sites in DNA, particularly the 0-6 position of guanine. The aforementioned mutagenesis result provided the first direct evidence for the mutagenic role of 0⁶-methylguanine and also demonstrated the high fidelity of in vitro replication of T7 under standard conditions. Measurements of reversion were made possible by modifying the encapsulation protocol so that very high DNA packaging efficiencies were routinely obtained. A novel application of the in vitro T7 system is to examine molecular mechanisms of host functions induced by DNA damage. and some experiments directed towards this goal were performed. Inducible reactivation of T7 phage damaged by alkylating agents or UV light was demonstrated and the phenomena were characterized with respect to the induction kinetics and dependence on host functions.