UV-C radiation induced conformational relaxation of pMTa4 DNA in Escherichia coli may be the cause of single strand breaks.

PURPOSE

The biological consequences of initial physicochemical events following exposure of DNA to germicidal (254 nm) ultraviolet C (UV-C) radiation are not fully understood despite progress that has been made. In particular the cause of UV-C induced single strand breaks is not known. This question has been addressed in the present investigation.

MATERIALS AND METHODS

A plasmid construct, pMTa4, was exposed to UV-C in vitro as well as in vivo after transforming the plasmid into a repair proficient wild type and repair deficient, recF, mutant of E. coli. Following UV exposure in vivo, the plasmid was isolated under repair non-permissive and permissive conditions. The plasmid isolate and the pure super-coiled closed circular (CC) topological form of the plasmid were analyzed by agarose gel electrophoresis. The dependence of UV-C induced damage and conformational changes on the dose of radiation as well as on the duration of post-irradiation repair incubations was observed. The influence of UV-C on hyperchromic change and intercalation of ethidium bromide into plasmid DNA were also recorded.

RESULTS

UV-C exposure of pMTa4 DNA in vitro and in vivo induced dose dependent, but sparsely placed, single strand breaks (SSB). While the wild type (AB1157) E. coli was able to repair SSB nearly completely under repair permissive condition, the recF (JC9239) mutant failed to do so. A dose-dependent relaxation of super-structure of CC form of pMTa4 DNA concomitant with enhanced ethidium bromide intercalation into the plasmid DNA was observed.

CONCLUSION

It is proposed that the conformational relaxation generated negative super-coiling strain on the DNA backbone of CC form of plasmid as well as exposed chemical bonds for hydrolytic cleavage. This might be the cause of the production of sparsely placed single strand breaks in pMTa4 upon exposure to low doses of UV-C.