Human alkyladenine DNA glycosylase employs a processive search for DNA damage

Abstract

DNA repair proteins conduct a genome-wide search to detect and repair sites of DNA damage wherever they occur. Human alkyladenine DNA glycosylase (AAG) is responsible for recognizing a variety of base lesions, including alkylated and deaminated purines, and initiating their repair via the base excision repair pathway. We have investigated the mechanism by which AAG locates sites of damage using an oligonucleotide substrate containing two sites of DNA damage. This substrate was designed so that AAG randomly binds to either of the two lesions. AAG-catalyzed base excision creates a repair intermediate, and the subsequent partitioning between dissociation and diffusion to the second site can be quantified from the rates of formation of the different products. Our results demonstrate that AAG has the ability to slide for short distances along DNA at physiological salt concentrations. The processivity of AAG decreases with increasing ionic strength to become fully distributive at high ionic strengths, suggesting that electrostatic interactions between the negatively charged DNA and the positively charged DNA binding surface are important for nonspecific DNA binding. Although the amino terminus of the protein is dispensable for glycosylase activity at a single site, we find that deletion of the 80 amino-terminal amino acids significantly decreases the processivity of AAG. These observations support the idea that diffusion on undamaged DNA contributes to the search for sites of DNA damage.

Figure 1

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.

Figure 2

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.

Figure 3

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).

Figure 4

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.

Figure 5

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).

Figure 6

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.

Figure 7

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.

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