The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U*A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U*A or U*G base pairs in a background of approximately 10(9) T*A or C*G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T*A and U*A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases.

The linking together of molecular fragments that bind to adjacent sites on an enzyme can lead to high affinity inhibitors. Ideally, this strategy would employ linkers that do not perturb the optimal binding geometries of the fragments and do not have excessive conformational flexibility that would increase the entropic penalty of binding. In reality, these aims are seldom realized due to limitations in linker chemistry. Here we systematically explore the energetic and structural effects of rigid and flexible linkers on the binding of a fragment-based inhibitor of human uracil DNA glycosylase. Analysis of the free energies of binding in combination with co-crystal structures shows that the flexibility and strain of a given linker can have a significant impact on binding affinity even when the binding fragments are optimally positioned. Such effects are not apparent from inspection of structures and underscore the importance of linker optimization in fragment-based drug discovery efforts.

Uracil DNA glycosylase (UNG) is a powerful DNA repair enzyme that has been shown to stabilize a glycosyl cation reaction intermediate and a related tight binding inhibitor using electrostatic interactions with the +1 and −1, but not the +2, phosphodiester group of the single-stranded DNA substrate Ap 2+ Ap 1+ Up 1− ApA. These experimental results differed considerably from computational findings using duplex DNA, where the +2 phosphate was found to stabilize the transition state by ~5 kcal/mol, suggesting that UNG uses different catalytic strategies with single-stranded and doublestranded DNA substrates. In addition, the computational studies indicated that the conserved and positively charged His 148 (which hydrogen bonds to the +2 phosphate) destabilized the glycosyl cation intermediate by 6-8 kcal/mol through anticatalytic electrostatic interactions. To evaluate these interesting proposals, we measured the kinetic effects of neutral methylphosphonate (MeP) stereoisomers at the +1 and +2 positions of a 12-mer dsDNA substrate and also the catalytic contribution and ionization state of His 148. For MeP substitutions at the +1 position, single-turnover kinetic studies showed that the activation barrier was increased by 9.8 and 3.1 kcal/mol, corresponding to a stereoselectivity of nearly 40000-fold for the respective MeP isomers. Identical to the findings with ssDNA, MeP substitutions at the +2 position resulted in only small changes in the activation barrier (±0.3 kcal/mol), with little stereoselectivity (~4-fold). However, the H148A mutation destabilizes both the ground state and transition states by 2.4 and 4.3 kcal/mol, respectively. Thus, His 148 is catalytic because it stabilizes the transition state to a greater extent (1.9 kcal/mol) than the ground state. Heteronuclear NMR studies established that His 148 was neutral in the free enzyme at neutral pH, and in conformational exchange in a specific DNA complex containing uracil. We conclude that the +1 and +2 phosphate esters play identical catalytic roles in the reactions of single-stranded and double-stranded DNA substrates, and that His 148 serves a catalytic role by positioning the substrate and catalytic water, or by an environmental effect.The diverse functional groups of substrates can provide unique opportunities for enzymes to co-opt mechanisms of catalysis that may not be possible in the absence of these groups. One example is the polymeric and negatively charged DNA phosphate ester backbone that can provide a robust scaffold for electrostatically driven binding interactions for enzymes that recognize DNA substrates (1). Quite often, such interactions may involve noncovalent bonding at one or more phosphate sites distant from the site of chemical reaction, and the favorable binding energy is used to drive the substrate into a reactive state that would be otherwise energetically inaccessible. More remarkably, the DNA repair enzyme uracil DNA glycosylase