Kinetic mechanism for binding and flipping of damaged bases by DNA repair dealkylases

Abstract

DNA is susceptible to alkylation by endogenous metabolites and by exogenous reactive species and multiple pathways have evolved to detect and repair the sites of alkylation damage. The best characterized pathway employs DNA repair glycosylases to remove alkylated bases and initiate the multi‐step base excision repair pathway. In contrast, the AlkB family of direct repair enzymes use oxygen, iron(II) and 2‐oxoglutarate to catalyze the oxidative removal of alkyl groups from nucleobases and restore them to their natural structures in a single step. Structural work shows that base flipping is used to bring the alkylated base into proximity of the catalytic iron, but kinetic studies have been difficult and crucial information is lacking regarding the timing of the searching, flipping and chemical steps. We have used equilibrium binding and transient kinetic studies to define the minimal kinetic mechanism for AlkB enzymes. We compare this mechanism to that which is employed by DNA repair glycosylases. Interesting parallels between these different pathways have been uncovered that provide insight into the fundamental problem of how aberrant methyl groups can be detected in duplex DNA. However, there are also several differences that are relevant to pathway choice in cells where both pathways are operational.