RATE OF INCISION OF N-ACETYL-2-AMINOFLUORENE AND N-2-AMINOFLUORENE ADDUCTS BY UVRABC NUCLEASE IS ADDUCT-SPECIFIC AND SEQUENCE-SPECIFIC - COMPARISON OF THE RATES OF UVRABC NUCLEASE INCISION AND PROTEIN-DNA COMPLEX-FORMATION

The UvrABC nuclease, the nucleotide excision repair complex from Esche richia coli, is able to incise a variety of types of DNA damage and th e repair efficiency of this enzyme complex appears to be influenced by the structure of the damage and the sequence context within which the damage is positioned. In order to better establish these relationship s, we have constructed two DNA sequences each containing a site-specif ically positioned N-2-aminofluorene (AF) or N-acetyl-2-aminofluorene ( AAF) adduct and have determined both the kinetics of UvrABC nuclease i ncision and the kinetics of UvrABC nuclease-substrate complex formatio n. It is well established that these two adducts induce very different structures in the DNA and that these structures also depend on the se quence context. We have found that the rate of incision of both AAF- a nd AF-DNA adducts is significantly faster when they are positioned in the mutation hotspot NarI sequence (5-GGCGCC-3') than when located in a normal or non-NarI sequence (5'-GATGATA-3') and that the rate of i ncision for AAF-DNA adducts is faster that for AF adducts in both sequ ences. Most siginificantly, we find that the rate of UvrB and UvrBC-su bstrate complex formation correlates with the rate of UvrABC nuclease incision.