Are enzymatically produced single-strand breaks involved in UV-induced inactivation of plasmid DNA?

Abstract

pBR322 plasmid DNA was exposed to 254 nm UV radiation and examined for enzymatically produced single-strand break (sbb) and double-strand break (dsb) formation by treatment with an extract containing the proteins of Escherichia coli (AB1157 (uvrA + recA +) and AB2480 (uvrA − recA −)). Enzymatic conversion of the 254 nm-induced lesions into ssbs on treatment with an extract from AB1157 was observed, but not conversion into dsbs. The rate of enzymatic ssb formation in the AB1157 extract is initially higher than in the AB2480 extract, the sbb formation levels off leading to plateau values with increasing incubation time. The rate of ssb formation in the AB2480 extract is initially lower, but does not level off, and the ssb yield becomes larger at longer incubation times than that with the AB1157 extract. The biological inactivation of the plasmids was measured as a function of 254 nm fluence by transformation of E. coli AB1157 and AB2480. Inactivation with AB2480 is mainly due to a single photoproduct, a cyclobutane-type pyrimidine dimer, per DNA molecule. Inactivation with AB1157 occurs with a quantum yield which is virtually identical with that of the plateau values of enzymatic ssb formation, as measured by incubation in the AB1157 extract. A possible interpretation is that the formation of irreparable ssbs is the lethal step in the sequence of events leading to inactivation of plasmid DNA in the repair wild-type strain. The quantum yield of inactivation is 10–20 times smaller for transformation of AB1157 than for AB2480, indicating that enzymatic repair of photolesions of the plasmid occurs in AB1157.