UvrAB activity at a damaged DNA site: is unpaired DNA present?

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.