To study the activity of the Escherichia coli UvrA and UvrB nucleotide excision 
repair proteins during the formation of the pre-incision complex at a damaged 
DNA site, we used substrates with modifications around a single 
2-(acetylamino)fluorene (AAF) lesion. Based on the release of AAF-containing 
oligonucleotides from a single-stranded DNA circle, we conclude that during 
interaction with our substrates UvrAB introduces changes in DNA which are 
localized at the lesion and are limited to 1-3 bp. Since these changes might 
include a denaturation of DNA at the lesion site and, consequently, a bubble 
structure might be present in a pre-incision complex, we studied incision 
activity of UvrABC excinuclease on substrates with 1-4 unpaired bases next to an 
AAF adduct. Opening more than one base on either or both sides of the lesion 
caused a significant decrease in the incision activity of UvrABC, but did not 
change the position of the incision sites. We conclude that the UvrAB action 
leading to a pre-incision complex does not include the formation of a bubble 
intermediate generated by extensive denaturation of base pairs.