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. 2017 Jul 7;45(12):7226-7236.
doi: 10.1093/nar/gkx223.

Quaternary interactions and supercoiling modulate the cooperative DNA binding of AGT

Manana Melikishvili  1 , Michael G Fried  1
Affiliations

Quaternary interactions and supercoiling modulate the cooperative DNA binding of AGT

Manana Melikishvili et al. Nucleic Acids Res. .

Abstract

Human O6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in single-stranded and duplex DNAs. The search for these lesions, through a vast excess of competing, unmodified genomic DNA, is a mechanistic challenge that may limit the repair rate in vivo. Here, we examine influences of DNA secondary structure and twist on protein-protein interactions in cooperative AGT complexes formed on lesion-free DNAs that model the unmodified parts of the genome. We used a new approach to resolve nearest neighbor (nn) and long-range (lr) components from the ensemble-average cooperativity, ωave. We found that while nearest-neighbor contacts were significant, long-range interactions dominated cooperativity and this pattern held true whether the DNA was single-stranded or duplex. Experiments with single plasmid topoisomers showed that the average cooperativity was sensitive to DNA twist, and was strongest when the DNA was slightly underwound. This suggests that AGT proteins are optimally juxtaposed when the DNA is near its torsionally-relaxed state. Most striking was the decline of binding stoichiometry with linking number. As stoichiometry and affinity differences were not correlated, we interpret this as evidence that supercoiling occludes AGT binding sites. These features suggest that AGT's lesion-search distributes preferentially to sites containing torsionally-relaxed DNA, in vivo.

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Figures

Figure 1.
Figure 1.
Model of an AGT–DNA complex with single-stranded DNA. Left: end-view, oriented with protein N-termini toward the reader. Right: side-view with protein N-termini oriented toward the left. Subunits are numbered for reference. The repeating unit of this structure contains one molecule of AGT (colors) plus 4 nt of DNA (black). Coordinates were derived from the structure of Daniels et al. (21), as described by Adams et al. (24). This model features extensive contact between proteins n and n+3, but no direct contact between nearest neighbors.
Figure 2.
Figure 2.
Discrete complexes form when AGT binds single-stranded and duplex 13-mer DNAs. Electrophoretic mobility shift assays (EMSAs) were performed at 20 ± 1°C. Panel A: titration of single-stranded 13 nt DNA (2.1 nM) with AGT (ranging from 0 μM to 9.6 μM) to form the low-mobility cooperative complex. The equilibration buffer was 10 mM Tris (pH 7.6 at 20°C), 1 mM DTT, 100 mM KCl, 0.1 mg/ml BSA. Samples were resolved in a 15% native polyacrylamide gel cast and run as described. Band designations: F, free DNA; B, bound DNA. Panel B: Titration of unmodified double stranded 13 bp DNA (1.6 nM) with AGT (ranging from 0 μM to 9.5 μM). Reaction and electrophoresis conditions were as described for Panel A, above. Band designations: F, free DNA; B, bound DNA.
Figure 3.
Figure 3.
Sedimentation equilibrium (SE) analyses of samples containing AGT and 13mer DNAs. Panel A: data for binding to single-stranded DNA. Samples contained 2.2 μM DNA and 12.1 μM AGT. Radial scans taken at 20 000 rpm (formula image), 26 000 rpm (▪) and 35 000 rpm (♦) are shown with vertical offsets for clarity. The smooth curves correspond to a global fit of Equation (2) to a dataset including these scans and ones obtained at [AGT] = 18.2 μM. Panel B: data for binding to double-stranded DNA. Samples contained 2.0 μM DNA and 11.0 μM AGT. Radial scans taken at 20 000 rpm (formula image), 26 000 rpm (▪) and 35 000 rpm (♦) are shown with vertical offsets for clarity. The smooth curves correspond to global fits of Equation (2) to a dataset that includes these scans and ones obtained at [AGT] = 16.5 μM. For both analyses the small residuals, symmetrically-distributed about zero (upper panels) indicate that the cooperative nP + D ⇆ PnD model is compatible with the mass distributions of DNA. These analyses returned n = 2.96 ± 0.09 for single-stranded and 2.85 ± 0.07 for duplex 13-mer DNAs, respectively.
Figure 4.
Figure 4.
Determination of ensemble average binding affinities for 13 nt, 13 bp, 16 nt and 16 bp DNAs. DNAs (∼3 × 10−9 M) were titrated with AGT protein (0 ≤ [AGT] ≤ 5.2 × 10−5M) in buffer consisting of 10 mM Tris (pH 7.6), 1 mM DTT, 1 mM EDTA, 100 mM NaCl. Free and bound DNA species were resolved by native electrophoresis (EMSA) as described for Figure 2. Each dataset is derived from two or more independent titrations. The smooth curves are fits of Equation (5) to the data.
Figure 5.
Figure 5.
Electrophoretic resolution of purified topoisomers. Closed circular pUC19 DNA was relaxed with Escherichia coli topoisomerase I and resolved into constituent toposiomers by preparative electrophoresis, as described in ‘Materials and Methods’ section. Here individual topoisomers are shown resolved on a 1.4% agarose gel, stained with ethidium bromide and photographed with UV transillumination. The gel lanes are labeled S (source DNA prior to purification), and with linkage differences with respect to relaxed DNA (0 to −6). The positions of bands containing relaxed circular DNA (N) and closed circular DNA (CC) are indicated in the left margin.
Figure 6.
Figure 6.
SE analyses of representative AGT-pUC19 DNA mixtures. Samples were centrifuged to equilibrium at 3000 rpm and 4°C, as described in ‘Materials and Methods’ section. Equation (1) was used to fit radial absorbance distributions for samples containing ∼11 nM DNA and ∼8.5 μM AGT. Samples contained DNAs of ΔLk = −1 (formula image), of ΔLk = −2 (△) and ΔLk = −5 (♦). Absorbance values were offset vertically to improve visual clarity. The smooth curves correspond to fits of Equation (1) to these data. Small, symmetrically-distributed residuals (upper panels) indicate that the two-species model represented by Equation (1) was consistent with the mass distributions of DNA in these samples.
Figure 7.
Figure 7.
Dependence of saturating stoichiometry on linking difference. Samples contained individually purified pUC19 topoisomers (8–12 nM) and 24 μM AGT in buffer containing 10 mM Tris (pH 7.6), 1 mM DTT, 1 mM EDTA, 100 mM NaCl. Stoichiometries were inferred from the weight-average reduced molecular weights of AGT–DNA complexes, measured at SE, as described in the ‘Materials and Methods’ section.
Figure 8.
Figure 8.
AGT binding to isolated pUC19 DNA topoisomers. Binding densities (ν) were calculated from weight-average reduced molecular weights of AGT–DNA complexes, measured at SE, as described in ‘Materials and Methods’ section. Linking differences with respect to relaxed form DNA are indicated by the numbers near each curve. The smooth curves are fits of the long-chain version of the McGhee-von Hippel equation (Equation 4) to the data. Binding parameters (K, ω) returned by these analyses are given in Figure 9.
Figure 9.
Figure 9.
Dependence of K, ω and K•ω on linking difference, ΔLk. These parameters were obtained by fitting Equation (4) to the binding data shown in Figure 8. The error bars represent 95% confidence limits. The product K•ω is a measure of the ensemble-average affinity of an AGT monomer for its DNA substrate. The minimum of K for −5 < ΔLk < −3 indicates that protein–DNA contacts are marginally less stable on slightly-underwound DNA than they are on the relaxed form. The maximum of ω for −5 < ΔLk < −2 indicates that protein–protein contacts are most stable on slightly-underwound DNA.
Figure 10.
Figure 10.
Do shared DNA-backbone contacts mediate cooperative interaction? This view of our current model of the cooperative assembly ((24); see also Figure 1) emphasizes a portion of the AGT–DNA interface. Two protein nearest-neighbors (colored blue and light green) and a segment of the shared DNA surface are shown, as well as a small segment of a third protomer (green). DNA atoms are colored according to the CPK convention (oxygen, red; phosphorus, orange-yellow; nitrogen, light blue; carbon, gray). The residue numbers of amino acid side chains that share DNA contacts are indicated.
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