Kinetic Analysis of the Effect of N-Terminal Acetylation on Thymine DNA Glycosylase.

Abstract

Thymine DNA glycosylase (TDG) is tasked with initiating DNA base excision repair by recognizing and removing T, U, the chemotherapeutic 5-fluorouracil (5-FU), and many other oxidized and halogenated pyrimidine bases. TDG contains a long, unstructured N-terminus that contains four known sites of acetylation: lysine (K) residues 59, 83, 84, and 87. Here, K to glutamine (Q) mutants are used as acetyl-lysine (AcK) analogues to probe the effect of N-terminal acetylation on the kinetics of TDG. We find that mimicking acetylation affects neither the maximal single-turnover rate kmax nor the turnover rate kTO, indicating that the steps after initial binding, through chemistry and product release, are not affected. Under subsaturating conditions, however, acetylation changes the processing of U substrates. Subtle differences among AcK analogues are revealed with 5-FU in single-stranded DNA. We propose that the subtleties observed among the AcK analogues may be amplified on the genomic scale, leading to regulation of TDG activity. N-terminal acetylation, though, may also play a structural, rather than kinetic role in vivo.