MULTIPLY DAMAGED SITES IN DNA - INTERACTIONS WITH ESCHERICHIA-COLI ENDONUCLEASE-III AND ENDONUCLEASE-VIII

Bursts of free radicals produced by ionization of water in close vicin ity to DNA can produce clusters of opposed DNA lesions and these are t ermed multiply damaged sites (MDS). How MDS are processed by the Esche richia coli DNA glycosylases, endonuclease (endo) III and endo VIII, w hich recognize oxidized pyrimidines, is the subject of this study. Oli gonucleotide substrates were constructed containing a site of pyrimidi ne damage or an abasic (AP) site in close proximity to a single nucleo tide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion, Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand bre ak, although cleavage at position one 5' or 3' was reduced compared wi th cleavage at positions three or six 5' or 3', Neither endo III nor e ndo VIII was able to remove the base lesion when the gap was positione d 1 nt 5' or 3' in the opposite strand, Cleavage of the modified pyrim idine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA bre akage at the site of the base lesion was equivalent to or less when th e gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion, Gel mobility shift analysis of the binding of endo V III to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced b y an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed, These data show that process ing of oxidized pyrimidines by endos III and VIII was strongly influen ced by the position and type of lesion in the opposite strand, which c ould have a significant effect on the biological outcome of the MDS le sion.