Abstract
Based on the binding of the UvrAB complex to a promoter region in transcription open complexes (Ahn, B., and Grossman, L. (1996) J. Biol. Chem. 271, 21453-21461) and the requirement of a single-stranded region for UvrAB helicase activity, we examined the binding of UvrAB proteins to synthetic bubble or loop regions in duplex DNA and the role of these regions in translocation of the UvrAB complex as well as incision of DNA damage. We found that the UvrAB complex was able to bind to bubble and loop regions with an affinity similar to that for damaged DNA in the absence of RNAP. The preferential recognition and incision of damaged sites by the UvrAB complex was observed downstream of the bubble or loop region in the strand complementary to the strand along which the UvrAB complex translocates. These results imply that the bubble region generated in duplex DNA by RNAP serves as a preferred entry site for the translocation of the UvrAB complex, and that preferential binding and unidirectional translocation of the UvrAB complex predetermine where incision is to occur.
Highlights
UvrAB helicase activity, we examined the binding of UvrAB proteins to synthetic bubble or loop regions in duplex DNA and the role of these regions in translocation of the UvrAB complex as well as incision of DNA damage
We found that the UvrAB complex was able to bind to bubble and loop regions with an affinity similar to that for damaged DNA in the absence of RNAP
The preferential recognition and incision of damaged sites by the UvrAB complex was observed downstream of the bubble or loop region in the strand complementary to the strand along which the UvrAB complex translocates. These results imply that the bubble region generated in duplex DNA by RNAP serves as a preferred entry site for the translocation of the UvrAB complex, and that preferential binding and unidirectional translocation of the UvrAB complex predetermine where incision is to occur
Summary
We found that the UvrAB complex was able to bind to bubble and loop regions with an affinity similar to that for damaged DNA in the absence of RNAP. We report that the UvrAB complex binds to bubble and loop regions with an affinity similar to that for damaged DNA, and that preferential incision was observed in Binding Experiments—Apparent dissociation constants for UvrA or UvrAB binding to DNA substrate can be determined by measuring the extent of complex formation as a function of protein concentration.
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