Kinetic mechanism for binding and flipping of damaged bases by alkyladenine DNA glycosylase.

摘要

Alkyladenine DNA glycosylase (AAG) initiates the base excision repair pathway that repairs damage to single bases within DNA. AAG recognizes lesions caused by alkylation and deamination. AAG first locates the site of damage then excises the damaged base, leaving an abasic site in the DNA that is further processed by other repair proteins to complete the pathway. Although many substrates have been identified and there are high resolution structures, our understanding of the AAG mechanism remains incomplete. To investigate this further, the thermodynamics and kinetics of binding and base-flipping, along with structural conformational changes of AAG, were investigated. The thermodynamics of AAG binding to damaged and undamaged DNA was examined using fluorescence anisotropy. Surprisingly, this revealed that multiple proteins could bind with nanomolar affinity to short DNA oligonucleotides, which might be a common phenomenon for DNA repair enzymes. These results reveal the pitfalls of studying DNA binding by fluorescence anisotropy, since nonspecific binding dominates the changes in signal. The kinetic mechanism of the AAG reaction with 1,N6-ethenoadenine (epsilonA)-containing DNA was established, including binding, nucleotide flipping, base excision, and product release steps, by taking advantage of the natural fluorescence of the epsilonA lesion. We observed that the flipping step is fast and the equilibrium for flipping is highly favorable. This kinetic mechanism maximizes specificity between damaged and undamaged bases. To study possible conformational changes in AAG, we took two approaches. First, tyrosine residues in the active site pocket were mutated to tryptophans to serve as fluorescence reporters. We found Y127W and Y159W mutants had robust activity towards epsilonA. However, a full kinetic characterization revealed that these mutations have large effects on the rates and equilibria for flipping. This suggests these mutants will have limited utility in studying recognition and flipping of other damaged nucleotides. Secondly, preliminary experiments established the feasibility of using NMR to study AAG and provided evidence for extensive conformational changes that take place upon binding to DNA. These studies have provided a mechanistic framework that will facilitate future investigations into the role of conserved residues and the energetic basis for the discrimination between damaged and undamaged DNA.