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Raman Spectroscopy of Uracil DNA Glycosylase-DNA Complexes: Insights Into DNA Damage Recognition and Catalysis



J Dong, A C. Drohat, J T. Stivers, K W. Pankiewicz, P R. Carey


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 the DNA Raman bands, which can be attributed to an increased level of base stacking, with little perturbation in the vibrational modes of the DNA backbone. A specific complex between UDG and duplex DNA containing 2 -Β-fluoro-deoxyuridine shows similar increases in the level of DNA base stacking, but also a substrate-directed conformational change in UDG that is not observed with undamaged DNA, consistent with an induced-fit mechanism for damage site recognition. The similar increases in the level of DNA base stacking for the nonspecific and specific complexes suggest a common enzyme-induced distortion in the DNA, potentially DNA bending. The difference spectrum of the extrahelical uracil base in the substrate-analogue complexes reveals only 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 result is at variance with the conclusion from a recent crystal structure, in which the UDG active site significantly distorts the flipped-out pseudouradine 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 free uracil at neutral pH, and indicates that the uracil is anionic. This is consistent with recent NMR results, which established that the enzyme stabilizes the uracil anion leaving group by 3.4 pKa units compared to aqueous 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.
No. 43


DNA complexes, raman spectroscopy, uracil DNA glycosylase


Dong, J. , Drohat, A. , Stivers, J. , Pankiewicz, K. and Carey, P. (2000), Raman Spectroscopy of Uracil DNA Glycosylase-DNA Complexes: Insights Into DNA Damage Recognition and Catalysis, Biochemistry (Accessed May 20, 2024)


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Created September 30, 2000, Updated October 12, 2021