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

Abigail E Wolfe, Patrick J O'Brien

Index: Biochemistry 48(48) , 11357-69, (2009)

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Abstract

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.