FORMATION OF DNA-REPAIR INTERMEDIATES AND INCISION BY THE ATP-DEPENDENT UVRB-UVRC ENDONUCLEASE

The Escherichia coli UvrB and UvrC proteins play key roles in DNA dama ge processing and incisions during nucleotide excision repair. To stud y the DNA structural requirements and protein DNA intermediates formed during these processes, benzo[a]pyrene diol epoxide-damaged and struc ture-specific 50-base pair substrates were constructed, DNA fragments containing a preexisting 3' incision were rapidly and efficiently inci sed 5' to the adduct, Gel mobility shift assays indicated that this su bstrate supported UvrA dissociation from the UvrB-DNA complex, which l ed to efficient incision, Experiments with a DNA fragment containing a n internal noncomplementary 11-base region surrounding the benzo[a]pyr ene diol epoxide adduct indicated that UVrABC nuclease does not requir e fully duplexed DNA for binding and incision, In the absence of UvrA, UvrB (UvrC) bound to an 11-base noncomplementary region containing a 3' nick (Y substrate), forming a stable protein-DNA complex (K-d simil ar to 5-10 nM). Formation of this complex was absolutely dependent upo n UvrC, Addition to this complex of ATP, but not adenosine 5'-(beta,ga mma-iminotriphosphate) or adenosine 5'-(beta,gamma-methylene)triphosph ate, caused incision three or four nucleotides 5' to the double strand -single strand junction, The ATPase activity of native UvrB is activat ed upon interaction with UvrC and enhanced further by the addition of Y substrate, Incision of this Y structure occurs even without DNA dama ge, Thus the UvrBC complex is a structure-specific, ATP dependent endo nuclease.