Ionic strength affects the processivity of AAG. Processivity at the optimal pH of 6.1 (A) and at pH 7.5 (B) was determined at increasing ionic strength, as described in the methods. Both Δ80 (open symbols) and full-length AAG (closed symbols) were examined. The average value of 3-8 independent determinations is shown and the error bars indicate the standard deviation for each condition.

Design of a simple substrate to monitor processivity of a DNA repair glycosylase. (A) A 47-mer oligonucleotide duplex was prepared that contains two εA lesions and the lesion-containing strand is labeled at both 3′ and 5′ ends by fluorescein (Fl). The immediate sequence contexts of the two lesions are identical (underlined). (B) The expected products after AAG-catalyzed base excision and alkaline cleavage of the abasic product. Only the lesion-containing strand is shown and the asterisk indicates a fluorescein label.

Electrostatic surface potential of the AAG catalytic domain reveals a positively charged DNA binding surface. The crystal structure of the AAG·εA-DNA complex was used to generate this figure (1F4R; (31)). Electrostatic calculations were performed with Pymol on the protein alone (W.L. DeLano; http://www.pymol.org) and APBS ((58); using a plug-in written by M. Lerner; http://wwwpersonal.umich.edu/~mlerner/PyMOL/). A continuum from -2 (red) to +2 (blue) is shown. A view of the active site and bound DNA is on the left, illustrating the positively charged DNA binding surface, and on the right the molecule is rotated horizontally by 180° to show that the positively charged surface continues around the protein.

Ionic strength affects multiple turnover excision of εA at pH 6.1, but not at pH 7.5. The multiple turnover rate constants for Δ80 (open circles) and full-length AAG (open squares) were measured at the indicated ionic strength. The average of 3-8 replicates is shown and the error bars indicate one standard deviation from the mean. The solid lines shows the best fits to a cooperative model in which multiple sodium ions cause an increased rate of dissociation up to the threshold at which the rate of dissociation is greater than the rate constant for N-glycosidic bond cleavage (see Methods for details).

Our studies have focused on the repair of εA lesions. This lesion is thought to be the result of lipid peroxidation and it is found at low levels in human cells under normal growth conditions (28, 29). Since εA cannot hydrogen bond with any of the normal bases (Scheme 1), it is expected to present a relatively low barrier to nucleotide flipping. Indeed, it appears to bind more tightly than other substrates (5, 30). The crystal structure of AAG bound to an extrahelical εA lesion shows that it is readily accommodated in the active site pocket and the backbone amide of His136 donates a hydrogen bond to the N6 atom (31).

Single turnover excision of εA is independent of ionic strength and the two sites are equivalent under all of the conditions tested. Single turnover excision of εA at site 1 (open circles) and site 2 (open squares) were measured with 1 μM DNA and 3, 6, and 9 μM Δ80 AAG. The observed rate constants were independent of the concentration of AAG, so the average and standard deviation are shown (each data point represents at least 9 independent determinations of the rate constant). The rates of excision at both sites are the same within error and independent of ionic strength (k_max = 0.2 min^-1). These results are the same as was previously reported for excision of a single εA lesion from a similar sequence context using a ³²P-based glycosylase assay, suggesting that the activity of AAG is not affected by either of the fluorescein labels (5).

The multiple turnover processivity assay. (A) This representative gel compares reactions containing full-length or Δ80 AAG at pH 6.1 with 50-150 mM ionic strength. All reactions contained 2 μM oligonucleotide substrate and 20 nM enzyme. Three time points between 5 and 20% reaction were chosen for each reaction condition (the time varies between 0.1 and 12 hours, since the steady state rate is dependent upon ionic strength). (B-G) Reaction progress curves for products (fragments A & C) and intermediates (fragments AB & BC) are shown for each set of reactions. In this experiment each reaction was performed in triplicate and additional time points analyzed on additional gels are included. The average values for each condition are plotted and the error bars indicate the standard deviation. Panels B, D, & F show results obtained with Δ80 AAG and Panels C, E, & G show results obtained with full-length AAG. The ionic strength was 50 mM (Panels A & B), 150 mM (Panels C & D), and 300 mM (Panels E & G). The fraction processive was calculated from these data and from additional experiments to obtain the average F_p values that are shown in Figure 6A.

Characterization of the processivity substrate under single turnover conditions. A representative time course for AAG-catalyzed excision of εA with 35 nM oligonucleotide duplex and 350 nM Δ80 AAG at pH 6.1 and an ionic strength of 300 mM (see Methods for details). (A) Fluorescent scan of a 20% denaturing polyacrylamide gel showing increasing incubation times from left to right. The positions of the 47-mer substrate (ABC), 37-mer (BC), 34-mer (AB), 11-mer (C), and 9-mer (A) products are shown on the right (see Figure 1 for a schematic of the substrate and expected products resulting from N-glycosidic bond cleavage and hydroxide-catalyzed abasic site hydrolysis). (B) The amount of each labeled DNA is expressed as a fraction of the total fluorescence and the symbol legend is inset into the plot. Since the substrate contains two labels and the other species contain only a single label, the maximum for the product and intermediate bands is 0.5. The very similar maximal fractions observed for products and intermediates demonstrate that no correction is needed for either labeling efficiency or quantum yield of the 5′ and 3′-fluorescein labels. Furthermore, the almost identical rate constants indicate that both sites are recognized by AAG with equal efficiency.

The premise of the processivity assay is that an enzyme capable of diffusion along DNA will randomly bind to a site on the substrate, then diffuse along the DNA to locate one or the other lesion. Under multiple turnover conditions with excess substrate, most substrates will not have a protein bound. When the enzyme dissociates there is a low probability of rebinding the same DNA molecule. If the enzyme can diffuse along DNA and sample many binding sites prior to dissociation, then it may be able to excise both lesions before dissociating. We refer to the ability to remove multiple lesions in a single binding encounter as a processive mode of action. In the extreme case of 100% processive action, no intermediates corresponding to a single excision will be found. If AAG lacks the ability to diffuse along the DNA (i.e., dissociation into solution is much faster than translocation to the other site of damage), then intermediates corresponding to action at only a single site will accumulate at the same rate as the terminal fragments (enzymatic events E₁ and E₂; Figure 1). This extreme is referred to as distributive action. Depending on the distance between the target sites and the solution conditions, the behavior of the enzyme is expected to lie somewhere between these two extremes. We use the fraction processive (F_p) to define the fraction of enzymatic binding events that are processive versus the total number of processive and distributive events (Scheme 2; see Methods and Supporting Information). The average processivity can be determined by following many DNA binding events under steady state conditions (15).