title: Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping.
abstract: Nucleotide flipping is a common feature of DNA-modifying enzymes that allows access to target sites within duplex DNA. Structural studies have identified many intercalating amino acid side chains in a wide variety of enzymes, but the functional contribution of these intercalating residues is poorly understood. We used site-directed mutagenesis and transient kinetic approaches to dissect the energetic contribution of intercalation for human alkyladenine DNA glycosylase, an enzyme that initiates repair of alkylation damage. When AAG flips out a damaged nucleotide, the void in the duplex is filled by a conserved tyrosine (Y162). We find that tyrosine intercalation confers 140-fold stabilization of the extrahelical specific recognition complex, and that Y162 functions as a plug to slow the rate of unflipping by 6000-fold relative to the Y162A mutant. Surprisingly, mutation to the smaller alanine side chain increases the rate of nucleotide flipping by 50-fold relative to the wild-type enzyme. This provides evidence against the popular model that DNA intercalation accelerates nucleotide flipping. In the case of AAG, DNA intercalation contributes to the specific binding of a damaged nucleotide, but this enhanced specificity comes at the cost of reduced speed of nucleotide flipping.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Jenna M
authors.initials: JM
authors.lastname: Hendershot
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA pjobrien@umich.edu.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: O'Brien
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D009154:Mutation
chemical_list: C004981:1,N(6)-ethenoadenine; D004247:DNA; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D000225:Adenine