HYDROPHOBIC FORCES DOMINATE THE THERMODYNAMIC CHARACTERISTICS OF UVRA-DNA DAMAGE INTERACTIONS

The Escherichia coli DNA repair proteins UvrA, UvrB and UvrC work toge ther to recognize and incise DNA damage during the process of nucleoti de excision repair (NER). To gain an understanding of the damage recog nition properties of UvrA, we have used fluorescence spectroscopy to s tudy the thermodynamics of its interaction with a defined DNA substrat e containing a benzo[a]pyrene diol epoxide (BPDE) adduct. Oligonucleot ides containing a single site-specifically modified N-2-guanine (+)-tr ans-, (-)-trans-, (+)-cis-, or (-)-cis-BPDE adducts were ligated into 50-base-pair DNA fragments. All four stereoisomers of DNA-BPDE adducts show an excitation maximum at 350 nm and an emission maximum around 3 80 to 385 nm. Binding of UvrA to the BPDE-DNA adducts results in a fiv e to sevenfold fluorescence enhancement. Titration of the BPDE-adducte d DNA with UvrA was used to generate binding isotherms. The equilibriu m dissociation constants for UvrA binding to (+)-trans-, (-)-trans-, ( +)-cis-, and (-)-cis- BPDE adduct were: 7.4 +/- 1.9, 15.8 +/- 5.4, 11. 3 +/- 2.7 and 22.4 +/- 2.0 nM, respectively. There was a lar negative change In heat capacity Delta C-p,obs(o), (-3.3 kcal mol(-1) K-1) acco mpanied by a relatively unchanged Delta G(obs)(o) with temperature. Fu rthermore, varying the concentration of KCl showed that the number of ions released upon formation of UvrA-DNA complex is about 3.4, a relat ively small value compared to the contact size of UvrA with the substr ate. These data suggest that hydrophobic interactions are an important driving force for UvrA binding to BPDE-damaged DNA. (C) 1998 Academic Press