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

Kirpota OO, Endutkin AV, Ponomarenko MP, Ponomarenko PM, Zharkov DO, Nevinsky GA - Nucleic Acids Res. (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, Department of Molecular Biology, Novosibirsk State University, 2 Pirogova Street and SB RAS Institute of Cytology and Genetics, 10 Lavrentieva Avenue, Novosibirsk 630090, Russia.

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.

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Linear correlations between the experimentally measured KM 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 KM 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).
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Figure 7: Linear correlations between the experimentally measured KM 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 KM 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).

Mentions: For KM values, the maximal positive Q = 0.083 [Equation (5)] was found for the negative correlation with the average ΔG° (P38;[–10;+10]) of the analyzed DNA sequences (Figure 7A). The correlation was significant for both the learning set, each strand of ds ODN1–ODN6 (r = −0.832, α < 0.001), and the control set, ODN7–ODN12 (r = −0.806, α < 0.0025) (see ‘Materials and Methods’ section for a description of learning and control sets). For the pooled set of completely complementary ds ODN1–ODN12, the following linear regression was found (r = −0.462, α < 0.025):(8)The deviation of the experimental KM values from the values calculated using the ‘limiting stage’ approximation (Equations (2) and (7); ΔKM = KM – KM{e0–10…G…e0+10/e#–10…C…e#+10}) varied from −10.3 to 38.3 nM for ODN1–ODN12 and from −27 to 373 nM for partially mismatched ODN13–ODN21. Unlike in the case of kcat, a statistically significant correlation was found between these values and the number of mismatches for the set of all 21 ds ODNs (r = 0.814, α < 0.00001, data not shown). Therefore, a linearly additive contribution of disturbance of strand complementarity to the KM value exists. Using the data for ODN1–ODN6 and ODN13–ODN15, we have introduced a regression correcting Equation (2) for the number of mismatches (N≠):(9)The predicted KM values calculated using Equation (9) for all 21 ds ODNs are shown in Figure 7B. They are statistically significant both for the learning set combining ODN1–ODN6 and ODN13–ODN15 (r = 0.850, α < 0.005) and for the control set combining ODN7–ODN12 and ODN16–ODN21 (r = 0.862, α < 0.0005).Figure 7.


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

Kirpota OO, Endutkin AV, Ponomarenko MP, Ponomarenko PM, Zharkov DO, Nevinsky GA - Nucleic Acids Res. (2011)

Linear correlations between the experimentally measured KM 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 KM 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).
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Figure 7: Linear correlations between the experimentally measured KM 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 KM 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).
Mentions: For KM values, the maximal positive Q = 0.083 [Equation (5)] was found for the negative correlation with the average ΔG° (P38;[–10;+10]) of the analyzed DNA sequences (Figure 7A). The correlation was significant for both the learning set, each strand of ds ODN1–ODN6 (r = −0.832, α < 0.001), and the control set, ODN7–ODN12 (r = −0.806, α < 0.0025) (see ‘Materials and Methods’ section for a description of learning and control sets). For the pooled set of completely complementary ds ODN1–ODN12, the following linear regression was found (r = −0.462, α < 0.025):(8)The deviation of the experimental KM values from the values calculated using the ‘limiting stage’ approximation (Equations (2) and (7); ΔKM = KM – KM{e0–10…G…e0+10/e#–10…C…e#+10}) varied from −10.3 to 38.3 nM for ODN1–ODN12 and from −27 to 373 nM for partially mismatched ODN13–ODN21. Unlike in the case of kcat, a statistically significant correlation was found between these values and the number of mismatches for the set of all 21 ds ODNs (r = 0.814, α < 0.00001, data not shown). Therefore, a linearly additive contribution of disturbance of strand complementarity to the KM value exists. Using the data for ODN1–ODN6 and ODN13–ODN15, we have introduced a regression correcting Equation (2) for the number of mismatches (N≠):(9)The predicted KM values calculated using Equation (9) for all 21 ds ODNs are shown in Figure 7B. They are statistically significant both for the learning set combining ODN1–ODN6 and ODN13–ODN15 (r = 0.850, α < 0.005) and for the control set combining ODN7–ODN12 and ODN16–ODN21 (r = 0.862, α < 0.0005).Figure 7.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, Department of Molecular Biology, Novosibirsk State University, 2 Pirogova Street and SB RAS Institute of Cytology and Genetics, 10 Lavrentieva Avenue, Novosibirsk 630090, Russia.

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.

Show MeSH