The recognition by Escherichia coli Uvr nucleotide excision repair proteins of a 
variety of lesions with diverse chemical structures and the presence of helicase 
activity in the UvrAB complex which can displace short oligonucleotides annealed 
to single-stranded DNA led to a model in which this activity moves UvrAB along 
undamaged DNA to damaged sites where the lesion blocks further translocation and 
the protein-DNA pre-incision complex is formed. To evaluate this mechanism for 
damage recognition, we constructed substrates with oligonucleotides of different 
lengths annealed to single-stranded DNA circles and placed a single 
2-(acetylamino)fluorene (AAF) lesion either on the oligonucleotide or on the 
circle. For the substrates with no lesion, the UvrAB complex effectively 
displaced a 22-mer but not a 27-mer or longer fragments. The presence of AAF on 
the oligonucleotide significantly increased the release of the 27-mer but 
oligomers of 30 or longer were not separated. Placing the lesion on the circular 
strand did not block the release of the fragments. Instead, the releasing 
activity of UvrAB was stimulated and also depended on the length of the annealed 
oligonucleotide. These observations do not agree with the predictions of a 
damage recognition mechanism that depends on helicase-driven translocation. Most 
likely, the strand-separating activity of UvrAB is a consequence of local 
changes occurring during the formation of a DNA-protein pre-incision complex at 
the damaged site and is not due to translocation of the protein along undamaged 
DNA to locate a lesion.