Abstract
UvrB, the ultimate damage-binding protein in bacterial nucleotide excision repair is capable of binding a vast array of structurally unrelated lesions. A beta-hairpin structure in the protein plays an important role in damage-specific binding. In this paper we have monitored DNA conformational alterations in the UvrB-DNA complex, using the fluorescent adenine analogue 2-aminopurine. We show that binding of UvrB to a DNA fragment with cholesterol damage moves the base adjacent to the lesion at the 3' side into an extrahelical position. This extrahelical base is not accessible for acrylamide quenching, suggesting that it inserts into a pocket of the UvrB protein. Also the base opposite this flipped base is extruded from the DNA helix. The degree of solvent exposure of both residues varies with the type of cofactor (ADP/ATP) bound by UvrB. Fluorescence of the base adjacent to the damage is higher when UvrB is in the ADP-bound configuration, but concomitantly this UvrB-DNA complex is less stable. In the ATP-bound form the UvrB-DNA complex is very stable and in this configuration the base in the non-damaged strand is more exposed. Hairpin residue Tyr-95 is specifically involved in base flipping in the non-damaged strand. We present evidence that this conformational change in the non-damaged strand is important for 3' incision by UvrC.
Highlights
5th phosphodiester bond 3Ј to the damage [6, 7], which is followed by an incision at the 8th phosphodiester bond 5Ј to the damage [8]
When base stacking is reduced as a consequence of the presence of damage, flipping will be facilitated, leading to formation of a stable UvrB-DNA complex
The cholesterol damage used is directly coupled to the DNA backbone via a carbon-linker [9], and there is no ribose or base present at this position
Summary
Proteins and DNA Fragments—The plasmid expressing the UvrB mutant (Y92A/Y93A) has been described [22]. Gel Retardation Assay—The 5Ј terminally labeled DNA substrates (0.2 nM) were incubated with 1.25 nM UvrA and 100 nM UvrB in 10 l of Uvr-endo buffer for 10 min at 37 °C. The proteins (0.45 M UvrA, 3.75 M UvrB, and 0.17 g/l CK) were incubated with the DNA in 10 l of Uvr-endo buffer (without bovine serum albumin) for 10 min at 37 °C in the presence of 10 or 20 mM CP, after which they were loaded on a 3.5% native gel as described above. 2-Aminopurine Fluorescence Measurements— 60-l samples containing 0.5 M DNA, 0.45 M UvrA, 3.75 M UvrB, and 0.17 g/l CK were incubated in Uvr-endo buffer (without bovine serum albumin) for 10 min at 37 °C in the presence of 10 or 20 mM CP as indicated. For testing the influence of the cofactor on fluorescence, 3 mM ADP, ATP, or ATP␥S were added to the mixture (which initially contained 1 mM ATP) at different time points as indicated
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