Kinetic mechanism for the excision of hypoxanthine by Escherichia coli AlkA and evidence for binding to DNA ends

Abstract

The Escherichia coli 3-methyladenine DNA glycosylase II protein (AlkA) recognizes a broad range of oxidized and alkylated base lesions and catalyzes the hydrolysis of the N-glycosidic bond to initiate the base excision repair pathway. Although the enzyme was one of the first DNA repair glycosylases to be discovered more than 25 years ago and there are multiple crystal structures, the mechanism is poorly understood. Therefore, we have characterized the kinetic mechanism for the AlkA-catalyzed excision of the deaminated purine, hypoxanthine. The multiple-turnover glycosylase assays are consistent with Michaelis-Menten kinetics. However, under single-turnover conditions that are commonly employed for studying other DNA glycosylases, we observe an unusual biphasic protein saturation curve. Initially, the observed rate constant for excision increases with an increasing level of AlkA protein, but at higher protein concentrations, the rate constant decreases. This behavior can be most easily explained by tight binding to DNA ends and by crowding of multiple AlkA protamers on the DNA. Consistent with this model, crystal structures have shown the preferential binding of AlkA to DNA ends. By varying the position of the lesion, we identified an asymmetric substrate that does not show inhibition at higher concentrations of AlkA, and we performed pre-steady state and steady state kinetic analysis. Unlike the situation in other glycosylases, release of the abasic product is faster than N-glycosidic bond cleavage. Nevertheless, AlkA exhibits significant product inhibition under multiple-turnover conditions, and it binds approximately 10-fold more tightly to an abasic site than to a hypoxanthine lesion site. This tight binding could help protect abasic sites when the adaptive response to DNA alkylation is activated and very high levels of AlkA protein are present.

Figure 1

Titration with a tight-binding pyrrolidine inhibitor to determine the concentration of active AlkA. Experiments were performed using 1 µM 19mer I•T substrate (19u) with varying concentrations (from 0 to 400 nM) of 25mer pyrrolidine inhibitor (Y•T). The concentration of AlkA was 100 nM (●) and 200 nM (○). The fraction of active AlkA was determined by measuring the initial rate of product formation and plotting the relative rate (V_obs/V_max) versus the concentration of inhibitor. The average and standard deviation of two to four replicates are shown. This titration gives an average value of 0.57 ± 0.03 for the fraction of active AlkA, assuming a single monomer binds to each DNA (11).

Figure 2

Single-turnover excision of Hx by AlkA. (A) Representative time course for AlkA-catalyzed excision from I•T 19u substrate (20 nM) at saturating concentration of AlkA (1.8 µM). Reactions were performed in triplicate and the average value is shown (error bars indicate the standard deviation). The reaction progress curve was fit by the equation for a single exponential (see Materials and Methods for details). (B) Dependence of the single turnover rate constant on the concentration of AlkA for I•T DNA. The symmetric 25mer substrate (□), the upstream shortened 19u (●) and the downstream shortened 19d (○) substrates are shown. Each data point is the average of at least 3 independent determinations and the error bars indicate the standard deviation. (C) The data for the 19d substrate are replotted from B to show that this substrate also shows inhibition at high concentration of AlkA. The equations for a simple hyperbolic dependence (eq 3) and for the inhibitory site model (eq 4) are given in the Materials and Methods and the values obtained from these fits are summarized in Table 2.

Figure 3

Multiple-turnover glycosylase activity of AlkA. (A) Representative data for AlkA-catalyzed excision of Hx from 19u (●), 19d (○), and the symmetric 25mer (■) with 10 nM AlkA and 1000 nM DNA. (B) The concentration dependence is shown for 25 (■) and 19u (●) I•T substrates. Each data point is the average of at least 3 independent determinations and the error bars indicate one standard deviation. The lines indicate the best fit to the Michaelis-Menten equation (eq 5). The k_cat and K_M values are given in Table 2.

Figure 4

AlkA-catalyzed excision of Hx under conditions suitable for detecting a burst. (A) Reactions were performed with saturating concentration of I•T 19u DNA (3 µM) and 34 (●), 68 (○), and 140 nM (□) AlkA. Reaction rates were linear, indicating the absence of a burst. (B) The initial rates from panel A are plotted as a function of the concentration of AlkA. The line shows a linear fit and yields a k_cat value of 0.12 ± 0.01 min⁻¹ that is in reasonable agreement with the k_cat value of 0.092 min⁻¹ that was determined from a wider range of substrate concentrations under steady-state conditions (Table 2).

Figure 5

Inhibition of AlkA by undamaged and abasic-containing DNA. Reactions contained 100 nM I•T 19u DNA, 2 nM AlkA, and the indicated concentration of abasic or undamaged (A•T) 25mer DNA inhibitor. Lines indicate the best fits to the equation for competitive inhibition and give K_d values of 61 ± 9 nM for undamaged DNA (●) and 2.8 ± 0.3 nM for abasic product (○; see inset).

Figure 6

Structure of AlkA bound to the end of a duplex. The figure was rendered with Pymol and the coordinates are from the Protein database (3CWT; (12)). This crystal form contains two oligonucleotides and four AlkA monomers per asymmetric unit, but only one AlkA-DNA interaction is shown. AlkA interacts predominantly with the strand that donates the 5’ end of the DNA. Arrows indicate the nucleotide position relative to the 5’end. Position 7 corresponds to the position of the lesion in the 19u substrate, and this lies on the opposite face of the DNA from a protein bound to the 5’ end. Position 13 (not present in the crystallized 12mer oligonucleotide) is on the same face as the end-bound AlkA molecule, but is separated by approximately one turn of the helix. This suggests that more than one AlkA molecule would need to bind to the 25mer symmetric substrate before the lesion site (position 13) would experience interference.

Figure 7

Models for inhibition of repair by binding of multiple AlkA molecules. If more than one AlkA (grey spheres) is bound ([AlkA] >> [DNA]), then not all sites can be sampled by the active site and the position relative to the end determines whether or not a damaged base (rectangle) is engaged productively (). If there is limiting amount of AlkA ([AlkA]<<[DNA]), then there will be an ensemble of DNA with AlkA bound at different binding sites across the DNA and every site can be sampled. For simplicity, only a few possible bound states are shown.

Figure 8

Minimal kinetic mechanism for the recognition and excision of hypoxanthine by AlkA. The binding and flipping steps are assumed to be in rapid equilibrium, as has been seen for other glycosylases (32). The absence of a burst indicates that the release of Hx (B) and abasic DNA (AP) products are not rate limiting. However, AlkA shows potent inhibition by abasic sites. The rate of N-glycosidic bond cleavage must be at least as fast as the observed rate constant for the single turnover reaction, however it could be significantly faster if the flipping equilibrium is unfavorable (37).

Scheme 1

Model to explain inhibition by multiple molecules of AlkA