CONFORMATION OF PLASMID DNA FROM ESCHERICHIA-COLI DEFICIENT IN THE REPAIR SYSTEMS PROTECTING DNA FROM 8-OXYGUANINE LESIONS

8-Oxyguanine (8ohG) is a major oxidation product of guanine and a biom arker of oxidative stress in mammal. We have attempted to estimate the level of 8ohG residues in plasmid DNA (pGW2123 and pBR322) grown in v arious bacterial strains (fpg, mutY, or mutT, plus mutT fpg and mutT m utY double mutants) differing in the system protecting cells against t he mutagenic effects of 8ohG in DNA. The method was based on digestion of plasmid DNA with Fpg, and agarose gel electrophoresis. Fpg convert s pDNA from covalently closed circular to the open circular (ccc --> c c) form of pDNA when there is at least one 8ohG, or apurinic site, per ccc pDNA molecule. It was found that: i) the content of 8ohG in pDNA grown in any of the tested bacteria is below one 8ohG per 10(4) base p airs; ii) a substantial part of pGW2123 is isolated from the bacteria in the oc form; iii) the ratio of oc/ccc in pGW2123 depends on the bac terial host and is the lowest when the plasmid was harvested from mutY - deficient cells; iv) pBR322, unlike pGW2123, is isolated predominant ly in the ccc form; and v) of the pBR322 grown in the tested bacteria apparently the most resistant to Fpg digestion was pBR322 grown in the mutY strain. It is proposed that this reflects the compact structure of pDNAs when they are grown in bacteria deficient in mutY gene produc t.