PMID:16282327
| Citation |
Malta, E, Moolenaar, GF and Goosen, N (2006) Base flipping in nucleotide excision repair. J. Biol. Chem. 281:2184-94 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1074/jbc.M508901200 |
| Keywords |
2-Aminopurine/chemistry; Adenosine Diphosphate/chemistry; Adenosine Triphosphate/chemistry; Base Sequence; DNA/chemistry; DNA Damage; DNA Helicases/chemistry; DNA Helicases/metabolism; DNA Repair; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Hydrolysis; Microscopy, Fluorescence; Molecular Conformation; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; Protein Binding; Protein Structure, Secondary; Sequence Homology, Nucleic Acid; Solvents/chemistry; Spectrometry, Fluorescence; Tyrosine/chemistry |
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