Linear free energy correlations for enzymatic base flipping: how do damaged base pairs facilitate specific recognition?
To efficiently maintain their genomic integrity, DNA repair glycosylases must exhibit high catalytic specificity for their cognate damaged bases using an extrahelical recognition mechanism. One possible contribution to specificity is the weak base pairing and inherent instability of damaged sites which may lead to increased extrahelicity of the damaged base and enhanced recognition of these sites. This model predicts that the binding affinity of the enzyme should increase as the thermodynamic stability of the lesion base pair decreases, because less work is required to extrude the base into its active site. We have tested this hypothesis with uracil DNA glycosylase (UDG) by constructing a series of DNA duplexes containing a single uracil (U) opposite a variety of bases (X) that formed from zero to three hydrogen bonds with U. Linear free energy (LFE) relationships were observed that correlated UDG binding affinity with the entropy and enthalpy of duplex melting, and the dynamic accessibility of the damaged site to chemical oxidation. These LFEs indicate that the increased conformational freedom of the damaged site brought about by enthalpic destabilization of the base pair promotes the formation of extrahelical states that enhance specific recognition by as much as 3000-fold. However, given the small stability differences between normal base pairs and U.A or U.G base pairs, relative base pair stability contributes little to the >10(6)-fold discrimination of UDG for uracil sites in cellular DNA. In contrast, the intrinsic instability of other more egregious DNA lesions may contribute significantly to the specificity of other DNA repair enzymes that bind to extrahelical bases.
Krosky
Daniel J
DJ
Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
Schwarz
Frederick P
FP
Stivers
James T
JT
D020029:Base Pairing; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry*; D004260:DNA Repair; D003853:Deoxyribonucleosides / Q000737:chemistry*; D004795:Enzyme Stability; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry*; D011196:Potassium Permanganate / Q000737:chemistry; D011485:Protein Binding; D013816:Thermodynamics; D013873:Thionucleotides / Q000737:chemistry; D051981:Uracil-DNA Glycosidase
D003853:Deoxyribonucleosides; D009692:Nucleic Acid Heteroduplexes; D013873:Thionucleotides; D011196:Potassium Permanganate; D004247:DNA; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase