Raman spectroscopy of uracil DNA glycosylase-DNA complexes: Insights into DNA damage recognition and catalysis

Using off-resonance Raman spectroscopy, we have examined each complex along the catalytic pathway of the DNA repair enzyme uracil DNA glycosylase (UDG ). The binding of undamaged DNA to UDG results in decreased intensity of th e DNA Raman bands, which can be attributed to an increased level of base st acking, with little perturbation in the vibrational modes of the DNA backbo ne. A specific complex between UDG and duplex DNA containing 2'-beta -fluor odeoxyuridine shows similar increases in the level of DNA base stacking, bu t also a substrate-directed conformational change in UDG that is not observ ed with undamaged DNA, consistent with an induced-fit mechanism for damage site recognition. The similar increases in the level of DNA base stacking f or the nonspecific and specific complexes suggest a common enzyme-induced d istortion in the DNA, potentially DNA bending. The difference spectrum of t he extrahelical uracil base in the substrate-analogue complexes reveals onl y a small electron density reorganization in the uracil ring for the ground state complex, but large 34 cm(-1) downshifts in the carbonyl normal modes . Thus, UDG activates the uracil ring in the ground state mainly through H bonds to its C=O groups, without destroying its quasi-aromaticity. This res ult is at variance with the conclusion from a recent crystal structure, in which the UDG active site significantly distorts the flipped-out pseudourid ine analogue such that a change in hybridization at C1 occurs [Parikh, S. S ., et al, (2000) Proc. Natl. Acad Sci. USA 97, 5083]. The Raman vibrational signature of the bound uracil product differs significantly from that of f ree uracil at neutral pH, and indicates that the uracil is anionic. This is consistent with recent NMR results, which established that the enzyme stab ilizes the uracil anion leaving group by 3.4 pK(a) units compared to aqueou s solution, contributing significantly to catalysis, These observations are generally not apparent from the high-resolution crystal structures of UDG and its complexes with DNA; thus, Raman spectroscopy can provide unique and valuable insights into the nature of enzyme-DNA interactions.