Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase

Abstract

We have used a stepwise increase in ligand complexity approach to estimate the relative contributions of the nucleotide units of DNA containing 7,8-dihydro-8-oxoguanine (oxoG) to its total affinity for human 8-oxoguanine DNA glycosylase (OGG1) and construct thermodynamic models of the enzyme interaction with cognate and non-cognate DNA. Non-specific OGG1 interactions with 10-13 nt pairs within its DNA-binding cleft provides approximately 5 orders of magnitude of its affinity for DNA (ΔG° approximately -6.7 kcal/mol). The relative contribution of the oxoG unit of DNA (ΔG° approximately -3.3 kcal/mol) together with other specific interactions (ΔG° approximately -0.7 kcal/mol) provide approximately 3 orders of magnitude of the affinity. Formation of the Michaelis complex of OGG1 with the cognate DNA cannot account for the major part of the enzyme specificity, which lies in the k(cat) term instead; the rate increases by 6-7 orders of magnitude for cognate DNA as compared with non-cognate one. The k(cat) values for substrates of different sequences correlate with the DNA twist, while the K(M) values correlate with ΔG° of the DNA fragments surrounding the lesion (position from -6 to +6). The functions for predicting the K(M) and k(cat) values for different sequences containing oxoG were found.

Figure 1.

The scale of ‘fuzzy decision’ of Zadeh fuzzy logic (34) given by projection of the output of the mth statistical test on the nth data set (the level of statistical significance α_m,n) onto an interval [−1, 1] with the fuzzy decision threshold q_m,n = 0, assigned to the statistical threshold α_m,n = 0.05, separating the positive fuzzy decision q_m,n >0 in the case of a statistically significant result (α_m,n <0.05) from the negative fuzzy decision q_m,n <0 in the case of an insignificant result (α_m,n ≥0.05).

Figure 2.

Analysis of the inhibition type and estimation of K_i for d(pT)₁₀:d(pA)₁₀ in the reaction of oxoG excision from ds OG11 catalyzed by OGG1, using a Lineweaver–Burk plot. The inhibitor was used at 0 (line 1), 0.15 (line 2), 0.30 (line 3) and 0.45 mM (line 4).

Figure 3.

Dependencies of log K_i on the length (n) of ss (solid line) and ds (dashed line) d(pN)_n inhibitors of oxoG excision from ds OG11 by OGG1. Triangles, d(pT)_n; circles, d(pA)_n; crosses, d(pC)_n; squares, d(pT)_n:d(pA)_n.

Figure 4.

Thermodynamic model of the interaction of OGG1 with non-cognate DNA. ΔG° values characterizing various contacts between the enzyme and DNA containing a G base are shown. All types of non-specific additive interactions of the enzyme and two strands of non-specific DNA provide ΔG° = −6.7 kcal/mol of total binding energy.

Figure 5.

Thermodynamic model of the interaction of OGG1 with cognate DNA with an oxoG base. Strengthening of the enzyme contacts with the damaged and complementary strands of the cognate DNA is indicated. The estimated ΔΔG° value characterizing the net change in the interactions of all types between non-cognate and cognate DNA complexed with OGG1 is −4.3 ± 0.2 kcal/mol. The amino acid residues of OGG1 interacting with specific DNA are shown after (25).

Figure 6.

Linear correlations between the experimentally measured k_cat values and either (A) the twist angle averaged over −6 to +6 position of the ODNs [Equation (3)] as calculated by the ACTIVITY software or (B) the k_cat values predicted by Equations (1), (3) and (7). Open circle and dashed line, learning set: (A) each strand of ds ODN1–ODN6 (r = 0.862, α <0.0005); (B) ds ODN1–ODN12 (r = 0.869, α <0.00025). Closed circle and straight line, control set: (A) each strand of ds ODN7–ODN12 (r = 0.835, α <0.001); (B) mismatched ds ODN13–ODN21 (r = 0.667, α <0.05).

Figure 7.

Linear correlations between the experimentally measured K_M values and either (A) the Gibbs free energy averaged over −10 to +10 position of the ODNs [Equation (3)] as calculated by the ACTIVITY software or (B) the K_M values predicted by Equations (3), (8) and (9). Open circle and dashed line, learning set: (A) each strand of ds ODN1–ODN6 (r = –0.832, α <0.001); (B) ds ODN1–ODN6 and ODN13–ODN15 (r = 0.850, α <0.005). Closed circle and straight line, control set: (A) each strand of ds ODN7–ODN12 (r = −0.806, α <0.00025); (B) ds ODN7–ODN12 and ODN16–ODN21 (r = 0.862, α <0.0005). Dotted line, all data combined: (A) each strand of ds ODN1–ODN12 [r = −0.457, α <0.025; Equation (8)]; (B) ds ODN1–ODN21 (r = 0.806, α < 0.00001).