Coordination of the initial steps of base excision repair: Characterizing apurinic/apyrimidinic endonuclease 1 stimulation of thymine DNA glycosylase

Abstract

DNA can be damaged by a variety of endogenous and exogenous sources. Cells are equipped with DNA repair systems to maintain genomic integrity. One mechanism for repair is the base excision repair (BER) pathway, in which a damage specific DNA glycosylase recognizes and excises damaged or mispaired bases, producing an abasic (AP) site in the DNA. Many DNA glycosylases bind AP-DNA product with high affinity, and exhibit slow enzymatic turnover in vitro. BER then continues as AP endonuclease I (APE1) displaces the DNA glycosylase and nicks the phosphodiester backbone. The AP site is excised and the original nucleotide is restored by other BER enzymes. Thymine DNA glycosylase (TDG) recognizes and repairs G∙T mismatches, as well as other lesions, with a preference for bases paired with guanine and located at CpG sites. Like other DNA glycosylases, TDG binds tightly to its product, AP-DNA, preventing enzymatic turnover. The mechanism for coordinating the transfer of toxic AP intermediates between the glycosylase and APE1 in base excision repair is poorly understood. Like TDG, other glycosylases bind AP-DNA very tightly, and APE1 has been shown to stimulate their turnover and relieve inhibition. The exact mechanism for displacement was unknown for TDG, but our studies on the effect of APE1 on Kcat using steady state kinetics experiments and measurements of individual rate constants using stopped flow anisotropy provide much needed insight into this mechanism. We find TDG activity is dramatically increased for G∙T substrates in the presence of APE1. Also, the steady state activity of TDG is limited by slow product release as well as inhibition by AP-DNA, with the greatest effect observed in reactions where commitment to catalysis is low (i.e. G∙T reactions). Substrate dissociation rates, inhibition experiments, and steady state kinetics all provide evidence for this phenomenon. Observation that product release and product inhibition contribute to slow Kcat suggests APE1 increases TDG turnover using passive and active mechanisms. Thus, the characterization of APE1 stimulation of TDG has provided valuable insight into the coordination of the initial steps of BER and into CpG site repair.