Mechanism of Nucleotide Flipping by Human Alkyladenine DNA Glycosylase.

[摘要] Nucleotide flipping is the phenomenon whereby a nucleotide in DNA is rotated 180 degrees out of the double helix into an extrahelical position. This event is critical to many biological processes in which enzymes need access to a specific nucleotide, but the mechanism remains poorly understood. Human alkyladenine DNA glycosylase (AAG) is one of many enzymes that use nucleotide flipping to engage substrates. AAG initiates the base excision repair pathway by locating sites of damage and catalyzing the hydrolysis of the N-glycosidic bond to release the damaged base. Previous crystal structures of AAG in complex with DNA revealed the DNA is bent where a tyrosine (Y162) intercalates into the space left by the flipped-out damaged nucleotide. The kinetic mechanism of binding, nucleotide flipping, and base excision for wild-type and mutant forms of AAG was determined. Mutation of active site tyrosines to tryptophans revealed a new intermediate that provides selectivity between damaged and undamaged sites prior to nucleotide flipping. Additionally, the functional contributions of the intercalating residue (Y162) to each of the steps in the AAG catalytic mechanism were determined. This residue acts as a selectivity filter and slows nucleotide flipping. Mutating the intercalating tyrosine to tryptophan revealed that intercalation occurs early in the search for damage. Similar intercalating interactions are observed in all nucleotide flipping enzymes, but the identity of this residue varies. Therefore, a structure/function study of AAG was performed in which the intercalating tyrosine was mutated to numerous amino acids of varying sizes. The results showed that the rate constants for nucleotide flipping and unflipping are faster for non-aromatic mutants, and an aromatic residue is required to stabilize the initial recognition complex and the stable recognition complex. Together, these results provide new insight into the mechanism of AAG, and have implications for other enzymes that use nucleotide flipping for DNA repair or epigenetic DNA modification.

[发布机构] University of Michigan

[效力级别] Biological Chemistry

[关键词] Kinetic mechanism of a DNA repair protein;Biological Chemistry;Science;Biological Chemistry