Distinguishing Specific and Nonspecific Complexes of Alkyladenine DNA Glycosylase.

Human alkyladenine DNA glycosylase (AAG) recognizes many alkylated and deaminated purine lesions and excises them to initiate the base excision DNA repair pathway. AAG employs facilitated diffusion to rapidly scan nonspecific sites and locate rare sites of damage. Nonspecific DNA binding interactions are critical to the efficiency of this search for damage, but little is known about the binding footprint or the affinity of AAG for nonspecific sites. We used biochemical and biophysical approaches to characterize the binding of AAG to both undamaged and damaged DNA. Although fluorescence anisotropy is routinely used to study DNA binding, we found unexpected complexities in the data for binding of AAG to DNA. Systematic comparison of different fluorescent labels and different lengths of DNA allowed binding models to be distinguished and demonstrated that AAG can bind with high affinity and high density to nonspecific DNA. Fluorescein-labeled DNA gave the most complex behavior but also showed the greatest potential to distinguish specific and nonspecific binding modes. We suggest a unified model that is expected to apply to many DNA binding proteins that exhibit affinity for nonspecific DNA. Although AAG strongly prefers to excise lesions from duplex DNA, nonspecific binding is comparable for single- and double-stranded nonspecific sites. The electrostatically driven binding of AAG to small DNA sites (∼5 nucleotides of single-stranded and ∼6 base pairs of duplex) facilitates the search for DNA damage in chromosomal DNA, which is bound by nucleosomes and other proteins.

Substitution of active site tyrosines with tryptophan alters the free energy for nucleotide flipping by human alkyladenine DNA glycosylase.

Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N(6)-ethenoadenine (εA), within 4-fold of that of the wild-type enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and εA fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the εA fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the εA fluorescence. Although these mutations do not have large effects on the rate constant for excision of εA, there are dramatic effects on the rate constants for nucleotide flipping that result in 40-100-fold decreases in the flipping equilibrium relative to wild-type. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in more stable base pairs.

Kinetic mechanism for the flipping and excision of 1,N(6)-ethenoadenine by human alkyladenine DNA glycosylase.

Human alkyladenine DNA glycosylase initiates the repair of a wide variety of alkylated and deaminated purine lesions in DNA. In this study, we take advantage of the natural fluorescence of the 1,N(6)-ethenoadenosine (epsilonA) lesion and report a kinetic analysis of binding, nucleotide flipping, base excision, and product release. The transient changes in the fluorescence of epsilonA revealed the existence of two distinct complexes that are formed prior to the hydrolysis step. An initial recognition complex forms rapidly and is characterized by partial disruption of the stacking interactions of the lesioned base. Subsequently, a very stable extrahelical complex is formed in which the epsilonA lesion is strongly quenched by interactions in the AAG active site pocket. Our results indicate that DNA binding and base flipping take place on the millisecond to second time scale. N-Glycosidic bond cleavage is much slower, taking place on the minute time scale. A pulse-chase experiment was used to demonstrate that even for the tightly bound epsilonA substrate, the extrahelical complex is not fully committed to excision. Nevertheless, flipping of epsilonA is highly favorable, and we calculate that the equilibrium constant for flipping is approximately 1300. This kinetic mechanism has important biological implications. First, two-step binding provides multiple opportunities to discriminate between damaged and undamaged nucleotides. Second, a rapid equilibrium flipping mechanism maximizes specificity for damaged versus undamaged bases, since undamaged bases generally form stronger base pairs than damaged bases. Finally, the highly favorable equilibrium for flipping of epsilonA ensures that epsilonA removal is independent of sequence context and highly efficient despite the relatively slow rate of N-glycosidic bond hydrolysis.

Interaction of benzoate pyrimidine analogues with class 1A dihydroorotate dehydrogenase from Lactococcus lactis.

Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of dihydroorotate to orotate in the only redox reaction in pyrimidine biosynthesis. The pyrimidine binding sites are very similar in all structurally characterized DHODs, suggesting that the prospects for identifying a class-specific inhibitor directed against this site are poor. Nonetheless, two compounds that bind specifically to the Class 1A DHOD from Lactococcus lactis, 3,4-dihydroxybenzoate (3,4-diOHB) and 3,5-dihydroxybenzoate (3,5-diOHB), have been identified [Palfey et al. (2001) J. Med. Chem. 44, 2861-2864]. The mechanism of inhibitor binding to the Class 1A DHOD from L. lactis has now been studied in detail and is reported here. Titrations showed that 3,4-diOHB binds more tightly at higher pH, whereas the opposite is true for 3,5-diOHB. Isothermal titration calorimetry and absorbance spectroscopy showed that 3,4-diOHB ionizes to the phenolate upon binding to the enzyme, but 3,5-diOHB does not. The charge-transfer band that forms in the 3,4-diOHB complex allowed the kinetics of binding to be observed in stopped-flow experiments. Binding was slow enough to observe from pH 6 to pH 8 and was (minimally) a two-step process consisting of the rapid formation of a complex that isomerized to the final charge-transfer complex. Orotate and 3,5-diOHB bind too quickly to follow directly, but their dissociation kinetics were studied by competition and described adequately with a single step. Crystal structures of both inhibitor complexes were determined, showing that 3,5-diOHB binds in the same orientation as orotate. In contrast, 3,4-diOHB binds in a twisted orientation, enabling one of its phenolic oxygens to form a very strong hydrogen bond to an asparagine, thus stabilizing the phenolate and causing charge-transfer interactions with the pi-system of the flavin, resulting in a green color.