UvrB, the ultimate damage-binding protein in bacterial nucleotide excision repair is capable of binding a vast array of structurally unrelated lesions. A ␤-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 nondamaged 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.DNA is constantly being threatened by various damaging agents, which can be either exogenous (chemicals or irradiation) or endogenous (reactive metabolites). If left unrepaired the damage could lead to mutations or cell death and therefore several repair mechanisms have evolved to avoid these effects. One of these mechanisms, nucleotide excision repair (NER) 2 is characterized by the unique feature that it is able to recognize and repair a large variety of structural different damages (1). In bacteria, NER is initiated by three different proteins: UvrA, UvrB, and UvrC (reviewed in Refs. 2 and 3). First UvrA and UvrB form a complex in solution, which is able to search the DNA for possible damage (4). Once damage has been found, UvrA leaves the complex resulting in a UvrB-DNA preincision complex. Atomic force microscopy (5) and bandshift analysis (23) of this complex have shown that it contains two UvrB molecules, with one monomer bound to the damaged site and the second monomer more loosely associated. The role of this second UvrB subunit in the repair reaction, however, still needs to be determined. UvrC subsequently binds to the preincision complex, thereby releasing the loosely associated UvrB subunit (5, 23). Next UvrC will make the incisions in the DNA. First an incision is made at the 4 th or 5 th phosphodiester bond 3Ј to the damage (6, 7), which is then followed by an incision at the 8 th phosphodiester bond 5Ј to the damage (8). The 5Ј incision is often followed by an extra incision seven nucleotides 5Ј to the original 5Ј incision, which has been shown to be the result of recognition of the 5Ј nick by UvrB (9). The resulting oligonucleotide is removed by UvrD after which PolI fills i...

UvrB plays a key role in bacterial nucleotide excision repair. It is the ultimate damage-binding protein that interacts with both UvrA and UvrC. The oligomeric state of UvrB and the UvrAB complex have been subject of debate for a long time. Using fluorescence resonance energy transfer (FRET) between GFP and YFP fused to the C-terminal end of Escherichia coli UvrB, we unambiguously show that in solution two UvrB subunits bind to UvrA, most likely as part of a UvrA2B2 complex. This complex is most stable when both UvrA and UvrB are in the ATP-bound form. Analysis of a truncated form of UvrB shows that binding to UvrA promotes dimerization of the two C-terminal domain 4 regions of UvrB. The presence of undamaged DNA leads to dissociation of the UvrA2B2 complex, but when the ATPase site of UvrB is inactivated, the complex is trapped on the DNA. When the complex is bound to a damaged site, FRET between the two UvrB subunits could still be detected, but only as long as UvrA remains associated. Dissociation of UvrA from the damage-bound UvrB dimer leads to the reduction of the magnitude of the FRET signal, indicating that the domain 4 regions no longer interact. We propose that the UvrA-induced dimerization of the domain 4 regions serves to shield these domains from premature UvrC binding. Only after specific binding of the UvrB dimer to a damaged site and subsequent release of UvrA is the contact between the domain 4 regions broken, allowing recruitment of UvrC and subsequent incisions.

SUMMARY Efficient repair of UV-induced DNA damage requires the precise coordination of nucleotide excision repair (NER) with numerous other biological processes. To map this crosstalk, we generated a differential genetic interaction map centered on quantitative growth measurements of >45,000 double mutants before and after different doses of UV radiation. Integration of genetic data with physical interaction networks identified a global map of 89 UV-induced functional interactions amongst 62 protein complexes, including a number of links between the RSC complex and several NER factors. We show that RSC is recruited to both silenced and transcribed loci following UV damage where it facilitates efficient repair by promoting nucleosome remodeling. Finally, a comparison of the response to high versus low levels of UV shows that the degree of genetic rewiring correlates with dose of UV and reveals a network of dose-specific interactions. This study makes available a large resource of UV-induced interactions, and it illustrates a methodology for identifying dose-dependent interactions based on quantitative shifts in genetic networks.

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