THE ACTUAL INCISION DETERMINES THE EFFICIENCY OF REPAIR OF CISPLATIN-DAMAGED DNA BY THE ESCHERICHIA-COLI UVRABC ENDONUCLEASE

The UvrABC endonuclease from Escherichia coli repairs a broad spectrum of DNA lesions with variable efficiencies. The effectiveness of repai r is influenced by the nature of the lesion, the local DNA sequence, a nd/or the topology of the DNA. To get a better understanding of the as pects of this multistep repair reaction that determine the effectivene ss of repair, we compared the incision efficiencies of linear DNA frag ments containing either a site-specific cis-[Pt(NH3)(2){d(GpG)-N7(1),- N7(2)}] or a cis-[Pt(NH3)(2)[d(GpCpG)-N7(1),-N7(3)}] adduct. Overall t he DNA with the cis-PtGG adduct was incised about 3.5 times more effic iently than the cis-Pt.GCG-containing DNA. The rate of UvrB-DNA preinc ision complex formation for both lesions was similar and high in relat ion to the incision. DNase I footprints, however, showed that the loca l structure of the two preincision complexes is different. An assay wa s developed to measure the binding of UvrC to the preincision complexe s and it was found that the binding rate of UvrC to the more slowly in cised cis-Pt.GCG preincision complex was higher than to the cis-Pt.GG preincision complex. This most likely reflects a qualitative differenc e in preincision complex structures. For both lesions the binding of U vrC to the preincision complex was fast compared to the kinetics of ac tual incision. Apparently, direct incision of cisplatin damage require s an additional conformational change after the binding of UvrC.