PHOTOLESIONS AND BIOLOGICAL INACTIVATION OF PLASMID DNA ON 254 NM IRRADIATION AND COMPARISON WITH 193 NM LASER IRRADIATION

Plasmid pTZ18R and calf thymus DNA in aerated neutral aqueous solution were irradiated by continuous 254 nm light. The quantum yields are PH I(ssb) = 4.0 x 10(-5) and PHI(dsb) = 1.4 x 10(-6) for single- and doub le-strand break formation, respectively, PHI(br) = 2.3 x 10(-5) for ba se release, PHI(dn) = 2.1 x 10(-3) for destruction of nucleotides, and PHI(icl) almost-equal-to PHI(lds) almost-equal-to 1 x 10(-6) for inte rstrand cross links and locally denatured sites, respectively. The pre sence of Tris-HCl/ethylenediamineletraacetic acid (10:1, pH 7.5) buffe r strongly reduces PHI(ssb). The corresponding PHI values, obtained on employing pulsed 193 nm laser irradiation, are much larger than those using lambda(irr) = 254 nm. This is ascribed to a contribution of che mical reactions induced by photoionization, which is absent for 254 nm ir-radiation. The quantum yields of inactivation of plasmid DNA (lamb da(irr) = 254 nm) were measured by transformation of the Escherichia c oli strains AB1157 (wild type), PHI(ina)(1157) = 1.6 x 10(-4), AB1886 (uvr-), PHI(ina)(1886) = 4.2 x 10(-4), AB2463 (rec-), PHI(ina)(2463) = 4.1 x 10(-4) and AB2480 (uvr- rec-), PHI(ina)(2480) = 3.1 x 10(-3). T he quantum yields of inactivation of plasmid DNA are compared with tho se of the four E. coli strains (denoted as chromosomal DNA inactivatio n) obtained from the literature. The results for E. coli strain AB2480 show that the chromosomal DNA and the plasmid DNA are both inactivate d by a single pyrimidine photodimer per genome. With the E. coli strai n AB2463 inactivation of plasmid and chromosomal DNA is the same for t he same total damage per genome and is almost-equal-to 10 times smalle r than for AB2480. This is explained by photodimer repair in chromosom al and plasmid DNA and by the absence of dsb repair in both cases. In the repair wild-type strain AB1157, inactivation of the plasmid DNA is roughly 100 times higher than that of the chromosomal DNA. We postula te that a portion of this difference is due to repair of dsb by the re cA system in chromosomal DNA and that such repair does not take place in the plasmid DNA. The biological results from 254 nm irradiation are compared with those from 193 nm laser irradiation.