DNA repair enzymes recognize and remove damaged bases that are embedded in the duplex. To gain access, most enzymes use nucleotide flipping , whereby the target nucleotide is rotated 180 degree into the active site. In human alkyladenine DNA glycosylase (AAG), the enzyme that initiates base excision repair of alkylated bases, the flipped-out nucleotide is stabilized by intercalation of the side chain of tyrosine 162 that replaces the lesion nucleobase. Previous kinetic studies provided evidence for the formation of a transient complex that precedes the stable flipped-out complex, but it is not clear how this complex differs from nonspecific complexes. We used site-directed mutagenesis and transient-kinetic approaches to investigate the timing of Y162 intercalation for AAG. The tryptophan substitution (Y162W) appeared to be conservative, because the mutant protein retained a highly favorable equilibrium constant for flipping the 1,N6-ethenoadenine (εA) lesion and the rate of N-glycosidic bond cleavage was identical to that of the wild-type enzyme. We assigned the tryptophan fluorescence signal from Y162W by removing two native tryptophan residues (W270A/W284A). Stopped-flow experiments then demonstrated that the change in tryptophan fluorescence of the Y162W mutant is extremely rapid upon binding to either damaged or undamaged DNA, much faster than the lesion-recognition and nucleotide-flipping steps that were independently determined by monitoring the εA fluorescence. These observations suggest that intercalation by this aromatic residue is one of the earliest steps in the search for DNA damage, and that this interaction is important for the progression of AAG from nonspecific searching to specific-recognition complexes.
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