Saturation of the single-turnover excision of Hx by AAG. Single-turnover reactions were performed with 20 nM DNA and the concentration of AAG was varied between 20 and 1000 nM. At each concentration of AAG the entire time course was followed and fit by a single exponential (Equation 3; see the Materials and Methods for details). The ionic strength in this experiment was 120 mM, and the average and standard deviation of duplicate reactions is shown. The results indicate that the K_1/2 for binding is ≤ 20 nM (Equation 4).

Equilibrium inhibition of AAG by its abasic DNA product. The relative affinity for substrate and inhibitor was determined for both full-length (●) and Δ80 AAG (■) by measuring the initial rate of product formation for mixtures of abasic product and substrate and plotting the relative activity (V_obs/V_max) versus the ratio of inhibitor to substrate. The concentration of AAG was 25 nM and the total concentration of DNA was maintained at 500 nM. The concentration of abasic DNA was varied from none to 450 nM. Reactions were performed in triplicate and the error bars indicate the standard deviation from the mean. The lines indicate the best fit of Equation 8 to the data (see Materials and Methods) and yield K_i/K_M ratios of 1.7 ± 0.2 for the full-length and 3.6 ± 0.2 for Δ80 AAG.

AAG exhibits burst kinetics. Reactions were carried out under our standard low ionic strength conditions (42 mM) with 1 µM 25mer substrate and 30 (●), 120 (■), or 300 (▲) nM full-length AAG. The reactions were performed in triplicate and the average and standard deviation are shown (in many cases the standard deviation is smaller than the symbols). The burst amplitude and steady state velocity were obtained from nonlinear least square fits to the data using Equation 5 (See Material and Methods for details). Both the burst amplitude and steady state velocity were linearly dependent upon the concentration of AAG, corresponding to a stoichiometric burst (see Supporting Information).

Model for the stimulation of AAG by APE1. A simplified mechanism is presented to emphasize the role of APE1 in stimulating the reaction of AAG. Although most lesions will be encountered by a nonspecifically bound protein that is diffusing along the duplex (25), only a single binding step is shown. Similarly, a single step is shown for the excision of Hx, but it is known that N-glycosidic bond cleavage is preceded by an unfavorable flipping equilibrium and therefore the maximal single-turnover reaction includes both the nucleotide flipping and N-glycosidic bond cleavage steps (7). According to our results, the release of the Hx product (X) is shown as a rapid step. Two possible pathways for dissociation of AAG and association of APE1 are shown. In the upper pathway AAG first dissociates and then APE1 binds. In the lower pathway APE1 binds to the AAG•abasic DNA complex and somehow increases the rate constant for dissociation of AAG. Our results demonstrate that the lower pathway is preferred for both full-length and truncated AAG under the conditions tested.

Stability of full-length AAG in the absence of DNA. AAG (20 nM) was incubated at pH 7.0 and 37 °C. The ionic strength was adjusted with sodium chloride to be low (42 mM, circles) or high (120 mM, squares). Incubations contained no additional proteins (open symbols) or were supplemented with 0.1 mg/mL BSA (closed symbols). After the indicated incubation time the multiple-turnover glycosylase activity was measured as described in the Materials and Methods. The average and standard deviation for triplicate reactions is shown. In the absence of BSA there was no detectable glycosylase activity, so only the limit of ≤1% is indicated. Single exponential fits to the activity of AAG in the absence of BSA gave half-lives of 10 and 40 minutes, respectively for low and high ionic strength. In the presence of BSA full glycosylase activity was retained for at least a day.

APE1 does not affect AAG-catalyzed single-turnover. Single-turnover reactions were carried out with either full-length (A & B) or Δ80 (C & D) AAG (1 µM) in excess over the 25mer DNA substrate (100 nM). These experiments were performed at 42 mM ionic strength (A & C) or at 120 mM ionic strength (B & D). The rates of reaction in the absence (○) and presence (●) of 2 µM APE1 were essentially identical in all cases. The lines indicate the non-linear least square fits of the equation for a single exponential (Equation 3). Representative data is shown for reactions that were carried out in duplicate and the error bars indicate the standard deviation. The rate constants obtained from this experiment were combined with additional experiments conducted under the same conditions and the average value is reported in Table 1.

The rate of dissociation of the abasic DNA product limits multiple-turnover excision by AAG in the absence of APE1. We took advantage of the faster rate of dissociation of shorter DNA substrates (Table 2) to independently test our model that dissociation from the abasic DNA product is the rate-limiting step in AAG-catalyzed multiple-turnover base excision. This experiment was carried out as described for Figure 7, except that the 17mer substrate was used in place of the 25mer substrate. Since faster multiple-turnover excision is observed for the shorter duplex, a lag phase is observed in the approach to steady state (○). The lag time obtained by extrapolation of the steady-state rate is 4 ± 1 minutes in good agreement with the expected lag time of 4 minutes [lag time = 1/(k_off 25mer + k_off 17mer)= 3.8 minutes]. In contrast, initiation of the reaction with a mixture of 25mer product and 17mer substrate gave rise to a burst phase (□). Reactions were performed in triplicate and the standard deviation is shown. The theoretical equations that were fit to the experimental data are described in the Material and Methods.

Addition of abasic DNA product is sufficient to eliminate the burst phase. The experimental design is given in part A & B. (A) AAG was preincubated with abasic 25mer product for 90 minutes (t1), after which 25mer substrate was added. Timepoints were taken during the second time period (t2) and the amount of abasic DNA product was determined with the standard DNA glycosylase assay. (B) As a reference reaction, enzyme was preincubated without DNA and the reaction was initiated by the addition of a mixture of 25mer abasic product and substrate to achieve identical conditions as in part A. The final concentrations after mixing were 50 nM AAG, 50 nM 25mer abasic DNA, and 1 uM 25mer substrate. In this representative experiment each reaction was performed in triplicate and the average and standard deviation for each time point is plotted in part C. A burst is observed when enzyme is added to the product/substrate mixture (□). No burst was observed when enzyme was first equilibrated with one equivalent of abasic DNA product (○). This confirms that the slow step in the multiple-turnover glycosylase reaction is release of the abasic DNA product, and that release of the Hx product must be as fast or faster than this step.

APE1 increases the rate of AAG-catalyzed multiple-turnover. The 25mer Hx-containing substrate (1 µM) was incubated with 50 nM of full-length (A & B) or Δ80 (C & D) AAG at low ionic strength (A & C) and at high ionic strength (B & D) in the absence (○) or presence (□) of 2 µM APE1. In cases for which greater than 20% of the substrate was converted to product, the closed symbols indicate the points that were used to determine the initial rate. It is apparent that the amino-terminal 80 amino acids of AAG are not required for the stimulation by APE1, because the upper panels (full-length AAG) are almost identical to the lower panels (Δ80 AAG). The linear fits to the initial rates provided k_cat values in the presence and absence of APE1 (Equation 1). This representative experiment was performed in triplicate, with the average and standard deviation (error bars) shown. The results from this experiment were combined with additional experiments to provide the rate constants that are reported in Table 1.

Satisfied that even dilute solutions of AAG were stable in our reaction conditions, we examined multiple-turnover base excision for reactions in which DNA substrate was in only slight excess to the enzyme. Unless otherwise indicated, we used a 25mer oligonucleotide duplex containing a central Hx•T mismatch (Scheme 1). This is much larger than the ∼10 base pairs footprint of AAG (13, 38). Under low ionic strength conditions (pH 7.0, 42 mM ionic strength) there is a rapid pre-steady state burst, followed by a much slower steady state rate (Figure 2). The concentration of AAG was varied at a constant concentration of DNA to evaluate whether the observed rapid phase of the reaction was a bona fide burst. The results demonstrate that the burst amplitude is linearly dependent upon the concentration of enzyme, whereas the burst rate constant is independent (Figure 2 & Supporting Information). The burst rate constant of ∼3 min⁻¹ was indistinguishable from the single-turnover rate constant of 2.8 ± 0.4 min⁻¹ (Figure 3 and Table 1). The burst amplitude was used to determine the concentration of active enzyme (see Materials and Methods). Similarly, the steady state rate of Hx excision is also linearly dependent upon the concentration of AAG (Figure 2 & Supporting Information). These data suggest that N- glycosidic bond cleavage reaction is relatively rapid and that it is followed by a much slower step that is required to regenerate free enzyme. Below we present evidence that release of the abasic DNA product is the rate-limiting step under the conditions employed.