Graduate Student University of Michigan Ann Arbor, Michigan, United States
Adam Thelen (University of Michigan)| Patrick O'Brien (University of Michigan)
Human DNA can be alkylated, oxidized, and deaminated through normal cellular processes or through exposure to exogenous agents. The DNA base excision repair pathway is tasked with correcting a variety of single-nucleotide sites of DNA damage. The pathway is initiated by DNA glycosylase enzymes, which hydrolytically cleave lesions from the genome at the N-glycosidic bond. Most glycosylases access their targets via a base flipping mechanism. Crystal structures of this flipped-out recognition complex consistently show that DNA is bent by varying degrees when bound by diverse glycosylases. This bending has been widely speculated to play a role in genome searching or in the optimization of the flipped-out recognition complex. Alkyladenine DNA glycosylase (AAG) recognizes a broad spectrum of alkylated and oxidized lesions and has been crystallized bound to DNA with a 20° bend. To clarify the role of DNA bending by AAG, the kinetics of bending were characterized using stopped flow FRET measurement of end-labeled oligonucleotides bound to AAG. These results were compared to the kinetics of base flipping measured via the fluorescence of lesion 1,N6-ethenoadenine. The rate constants for DNA bending and base flipping consistently aligned. Multiple tyrosine residues in the active site pocket of AAG were mutated to perturb the kinetics of base flipping. This resulted in equal changes to the kinetics of DNA bending, indicating that DNA bending does not precede base flipping. These findings suggest that DNA bending is not used for genome searching, but rather as a means to position or stabilize the flipped-out lesion.
Support or Funding Information
Work supported by a University of Michigan Department of Biological Chemistry graduate student award, Chemistry/Biology Interface Training Program fellowship from the NIGMS, National Institutes of Health Grants T32GM008597 (to A. Z. T.) and R01GM108022 (to P. J. O.), and National Science Foundation Grant MCB-1615586 (to P. J. O.).