Isolating contributions from intersegmental transfer to DNA searching by alkyladenine DNA glycosylase

Abstract

Large genomes pose a challenge to DNA repair pathways because rare sites of damage must be efficiently located from among a vast excess of undamaged sites. Human alkyladenine DNA glycosylase (AAG) employs nonspecific DNA binding interactions and facilitated diffusion to conduct a highly redundant search of adjacent sites. This ensures that every site is searched, but could be a detriment if the protein is trapped in a local segment of DNA. Intersegmental transfer between DNA segments that are transiently in close proximity provides an elegant solution that balances global and local searching processes. It has been difficult to detect intersegmental transfer experimentally; therefore, we developed biochemical assays that allowed us to observe and measure the rates of intersegmental transfer by AAG. AAG has a flexible amino terminus that tunes its affinity for nonspecific DNA, but we find that it is not required for intersegmental transfer. As AAG has only a single DNA binding site, this argues against the bridging model for intersegmental transfer. The rates of intersegmental transfer are strongly dependent on the salt concentration, supporting a jumping mechanism that involves microscopic dissociation and capture by a proximal DNA site. As many DNA-binding proteins have only a single binding site, jumping may be a common mechanism for intersegmental transfer.

Keywords: Base Excision Repair; DNA Glycosylase; DNA Repair; Enzyme Kinetics; Enzyme Mechanisms; Facilitated Diffusion; Intersegmental Transfer; Mutagenesis Mechanisms.

FIGURE 1.

Mechanisms of facilitated diffusion for searching DNA. The local search involves sliding and hopping, and this is highly redundant. To prevent becoming trapped in a small region, the local search must be balanced by long range events (three-dimensional diffusion or intersegmental transfer).

FIGURE 2.

Oligonucleotides used to monitor the diffusion of AAG. A, multiple turnover processivity assays monitor the partitioning between dissociation (k_off) and finding the second lesion (k_capture). One possible pathway is shown, but binding is random, and so there are two pathways for processive excision to form abasic sites (Ab) from the ethenoadenosine lesions (E). B, two-lesion and one-lesion oligonucleotide sequences (asterisk indicates the position of fluorescein labels).

FIGURE 3.

Steady state competition experiments to evaluate binding to PEG and single-strand DNA. A, if AAG is capable of binding to, and diffusing from, a 3′ extension, then there will be a larger number of productive encounters relative to DNA substrate lacking the extension and a larger value of k_cat/K_m will be observed. B, at low salt concentration AAG binding is irreversible, and the protein uses facilitated diffusion to search every site of a DNA molecule. The k_cat/K_m values simply reflect the association rate constant. C, at high salt concentration, AAG binding is fully reversible, and its glycosylase activity is distributive. D, the indicated substrates were competed against an equal concentration of 25-mer duplex with a central ϵA·T lesion, and the relative k_cat/K_m values are shown (mean ± S.D., n ≥ 3). The 100 mm data (white bars) indicate that AAG can productively transfer from single-strand DNA to duplex DNA but is unable to transfer from PEG to duplex DNA. The 1 m data (gray bars) provide an important control that confirms that the intrinsic reactivity is the same for all ϵA·T sites that were tested. Statistical significance for comparison of 1 m and 100 mm NaCl conditions was evaluated by Student's t test using GraphPad Prism (* denotes p ≤ 0.001).

FIGURE 4.

Processivity assays with PEG-tethered duplexes demonstrate that AAG is capable of intersegmental transfer. A, the steady state processivity of the previously characterized substrate (47E2F2) (11), in which two ϵA lesions are located 25 bp apart, is compared with the processivity of substrates in which the two ϵA lesions are on separate DNA molecules that are tethered with PEG linkers. The Na⁺ concentration was 200 mm. B, comparison of the salt dependence for the processivity of AAG with the PEG-10 substrate. C, the values of V/E for the experiments shown in panel B. Each value is the average ± S.D. (n > 3).

FIGURE 5.

Pre-steady state kinetics with two-lesion substrates. A, minimal steps involved in the AAG-catalyzed reaction for oligonucleotides with two ϵA lesion sites. The apparent rate constant for capture (k_capt) is dictated by the rate constant for transfer to the second site (k_trans) and ϵA excision (k_chem). B and C, burst experiments were performed with a 1:10 ratio of AAG to DNA substrate (47E2F2 or PEG-10), and the concentration of each species was calculated from the fraction of the total fluorescence determined by denaturing PAGE. Reactions were performed in triplicate, and the mean ± S.D. is plotted. Lines indicate the fits according to the irreversible model shown (see the supplemental material for the derivation of the equations; rate constants are compiled in Table 1). The total concentration of Na⁺ was either 50 mm (B) or 20 mm (C).

FIGURE 6.

Transfer to a new DNA molecule is promoted at higher DNA concentration. A, minimal kinetic mechanism for the AAG-catalyzed reaction on an oligonucleotide with two sites of damage. At dilute concentrations of DNA, the rate-limiting step is dissociation from the abasic product (k_off), but at higher concentrations of DNA, an intermolecular transfer step (k_trans) accelerates the overall rate of reaction. Multiple turnover processivity assays were performed at 50–150 mm Na⁺ using the 47E2F2 substrate. B, reaction velocities (mean ± S.D., n ≥ 3) were fit by linear regression (R² ≥ 0.94). C, the fraction processive was calculated as described under “Experimental Procedures” and analyzed by linear regression. The 50 and 100 mm NaCl conditions did not show a significant slope (p > 0.01; GraphPad Prism). At 150 mm NaCl, a modest, yet statistically significant, slope was observed (p < 0.0001), indicating that intersegmental transfer begins to compete with finding the second lesion. This additional pathway for transfer is illustrated by the dashed lines in panel A.

FIGURE 7.

Stimulation of AAG by nonspecific DNA. Multiple turnover reactions of the processivity substrate (47E2F2) were performed in the presence of increasing concentrations of 25-mer competitor DNA at different concentrations of NaCl. The open symbols correspond to a nonspecific 25-mer (25A·T), and the closed symbols correspond to a specific inhibitor (25D·T) DNA. A, the velocity increases with increasing concentration of nonspecific DNA, indicating that intersegmental transfer provides an alternative pathway to dissociation from the abasic product and that subsequent transfer from the nonspecific DNA is rapid. In contrast, the specific inhibitor has no effect on the reaction velocity. This can be explained by the fact that dissociation from the abasic analog is slow. B, the fraction processive is unchanged by increasing concentration of competitor at low salt (no significant slope, p > 0.01; GraphPad Prism). At 150 mm NaCl, a modest, yet statistically significant (p < 0.003), dependence is observed for both the nonspecific competitor and the specific inhibitor DNA. Values are the mean ± S.D. (n ≥ 3, except for the 8 μm 25A·T point for which n = 2).

FIGURE 8.

The amino terminus of AAG is not required for intersegmental transfer. A, AAG has a poorly conserved amino-terminal region (white) and a highly conserved carboxyl-terminal catalytic domain (black). B, the processivity of Δ80 AAG is not decreased by a PEG linker. Processivity measurements were performed at 115 mm NaCl as described for full-length AAG. C, multiple turnover glycosylase activity was measured for 47E2F2 with 50 mm Na⁺. The stimulation of multiple turnover glycosylase activity by high concentrations of DNA is similar for full-length and Δ80 AAG.

FIGURE 9.

Models for intermolecular/intersegmental transfer by DNA-binding proteins. Direct transfer between sites that are distant on the same DNA molecule is equivalent to direct transfer between two DNA molecules. A, the presence of two DNA binding domains (red and blue spheres) allows for a bridging intermediate in which two segments of DNA are simultaneously bound. This mechanism has traditionally been referred to as intersegmental transfer (1). Here we refer to this as the bridging mechanism. B, proteins with a single DNA binding site may exhibit intersegmental transfer without macroscopic release into bulk solvent provided that the probability of recapture is sufficiently high. This has been called “jumping” (28). The results for AAG are best explained by the jumping mechanism of intersegmental transfer.