Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic/apyrimidinic endonuclease from human placenta

Abstract

X-ray analysis of enzyme-DNA interactions is very informative in revealing molecular contacts, but provides neither quantitative estimates of the relative importance of these contacts nor information on the relative contributions of specific and nonspecific interactions to the total affinity of enzymes for specific DNA. A stepwise increase in the ligand complexity approach is used to estimate the relative contributions of virtually every nucleotide unit of synthetic DNA containing abasic sites to its affinity for apurinic/apyrimidinic endonuclease (APE1) from human placenta. It was found that APE1 interacts with 9-10 nt units or base pairs of single-stranded and double-stranded ribooligonucleotides and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleotide phosphate groups. Such nonspecific interactions of APE1 with nearly every nucleotide within its DNA-binding cleft provides up to seven orders of magnitude (DeltaG degrees approximately -8.7 to -9.0 kcal/mol) of the enzyme affinity for any DNA substrate. In contrast, interactions with the abasic site together with other specific APE1-DNA interactions provide only one order of magnitude (DeltaG degrees approximately -1.1 to -1.5 kcal/mol) of the total affinity of APE1 for specific DNA. We conclude that the enzyme's specificity for abasic sites in DNA is mostly due to a great increase (six to seven orders of magnitude) in the reaction rate with specific DNA, with formation of the Michaelis complex contributing to the substrate preference only marginally.

Figure 1

(a) Dependencies of the initial rate of APE1-catalyzed cleavage of [³H]AP DNA (3.5 U A₂₆₀/ml) on the concentration of ss d(pA)₁₀ (1) and ss d[(pT)₇pR(pT)₆] (2). The activity of APE1 in the absence of inhibitors was taken as 100%. (b) Lineweaver–Burk plot of the dependence of the initial rate of APE1-catalyzed accumulation of the acid-soluble short [³H]DNA product on the concentration of [³H]AP DNA at different concentrations of d(pT)₁₀: 0 μM (1), 1.6 μM (2) and 7.8 μM (3) d(pT)₁₀.

Figure 2

Dependencies of −logK_I on the length of inhibitor (n) for ss and ds deoxyribooligonucleotides (a) and ribooligonucleotides (b). (a) d(pT)_n (crosses), d(pA)_n (filled squares), d(pC)_n (open circles), d(pG)_n (triangles), d[(pF)_npT] (open squares), d(pT)_n·d(pA)_n (filled circles). (b) (pU)_n (crosses), (pC)_n (open circles), (pA)_n (filled squares), (pU)_n·(pA)_n (closed circles); the curves for d(pA)_n (crosses) and d(pT)_n·d(pA)_n (diamonds) are given for comparison.

Figure 3

Logarithmic dependencies of factor f for APE1 on the relative hydrophobicity of nucleotide bases of homo-d(pN)_n estimated from isocratic reverse phase chromatography of different nucleosides according to ref. (46). Extrapolation of the curve to zero hydrophobicity corresponding to orthophosphate gives an electrostatic factor e = 1.51.

Figure 4

Schematic structure of DNA binding site of APE1. The DNA-binding site of the enzyme consists of two sets of ten subsites each, but only one set of subsites interacting with the cleaved strand, shown in the figure, contains a specific subsite (‘0’ subsite) with increased affinity for one specific or nonspecific nucleotide unit of DNA. Lengthening of nonspecific d(pN)_n (1 ≤ n ≤ 10) leads to the formation of several alternative thermodynamically comparable complexes of these ODNs with different subsites on the enzyme.

Figure 5

Thermodynamic model of APE1 interactions with nonspecific DNA. For the enzyme subsites interacting with the cleaved strand, the ΔG° values characterizing their contacts with the d(pA)_n chain of d(pA)_n·d(pT)_n are given; for the subsites interacting with the noncleaved strand, the ΔG° values refer to their contacts with the d(pT)_n chain. All types of nonspecific additive interactions of APE1 with the d(pA)_n·d(pT)_n duplex provide ΔG° ∼ −7.96 kcal/mol.

Figure 6

(A) Schematic representation of contacts between APE1 and specific ds DNA revealed by X-ray crystallography (3,15). Arrows indicate interactions between the various amino acid residues and structural elements of DNA, assisting the sharp DNA kinking (see text for details). (B) Thermodynamic model of APE1 interactions with specific DNA, displaying ΔG° values characterizing different contacts and strengthening of some contacts in comparison with nonspecific DNA (see Figure 5). The total ΔΔG° value characterizing a change in all types of interactions upon transition from nonspecific to specific DNA can be estimated at −1.1 to −1.5 kcal/mol.