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

      Fitzgerald, Megan Elizabeth; Drohat, Alexander Clark (2011)
      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.
    • Finding the Needle in the Haystack: Characterization of the Catalytic and Binding Specificity of Thymine DNA Glycosylase (TDG)

      Morgan, Michael Thomas; Drohat, Alexander Clark (2011)
      Thymine DNA glycosylase (TDG) is a base excision repair enzyme responsible for the repair of G·T mispairs within a CpG sequence context. G·T frequently arises at these sites because of deamination of 5-methylcytosine (5-MeC), which occurs primarily at CpG sites. These mismatches are difficult to locate because they consist of two normally-occurring nucleotides. Our first study examined the mechanisms that TDG employs for catalytic specificity. Using uracil, thymine, and several 5-halouracils (5-FU, 5-ClU, and 5-BrU), we systematically increased the size of the C5 substituent (U<FU<ClU≤T<BrU) while altering the 5' base pair (defining CpG context) and changing the base opposing the target to adenine (analogous to a A:T base pair). The increasing size of C5 substituents results in greater degrees of context dependence in catalysis, likely due to a greater necessity for optimal base-flipping promoted by sequence-specific contacts. Notably, changing the pairing partner of the target base from guanine to adenine results in an approximate 18,000-fold decrease in activity against thymine. Next, the 2:1 binding complex observed in the TDG<sub>core</sub>:Ap-DNA crystal opened questions regarding binding stoichiometry and affinity for substrate and undamaged DNA. Using fluorescence anisotropy in equilibrium binding conditions accompanied by EMSAs, we determined that TDG is able to bind mismatches at nanomolar affinities with a second subunit binding at much higher concentrations. We used the non-hydrolysable substrate analogs U<super>F</super> and T<super>F</super> for these experiments to observe formation of the pre-catalytic complex. Using DynaFit, binding affinities were determined for mispairs G∙U<super>F</super> (K<sub>d1</sub>= 0.63 ± 0.16 nM) and G∙TF (K<sub>d1</sub>= 18 ± 3 nM), abasic DNA (Kd1= 1.4 ± 0.4 nM), an undamaged CpG site (K<sub>d1</sub>= 63 ± 10 nM), and undamaged non-specific DNA (K<sub>d1</sub>= 293 ± 64 nM). We concluded from these data that TDG is unlikely to bind in a 2:1 conformation in vivo. In another study, we found that N-terminal residues 56-110 significantly contribute to binding affinity and catalysis for damaged and undamaged DNA. The 56-110 region enhances binding of DNA containing G∙U<super>F</super> 64-fold, G∙T<super>F</super> 190-fold, and CpG sites 70-fold. These results extend our understanding of the specificities of the catalytic mechanism of TDG.