Limits...
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.

Show MeSH
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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113562&req=5

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.

Mentions: Based on the measurements of binding affinity of OGG1 for various ligands, we have constructed a model summarizing the contribution of different interactions to DNA binding by OGG1. As mentioned above, extrapolation of the log(Ki) dependencies to n = 1 gives a Ki value of 12 mM, which characterizes the interaction of the active site of OGG1 with one of the nucleotides of d(pN)n bound to the protein. From this, the ΔG° of one non-specific dNMP interaction with the active site of OGG1 can be estimated as −2.6 kcal/mol. Since 12 phosphate groups of one strand of ds d(pN)13 interact with OGG1 through weak, additive contacts, (ΔG° approximately −0.34 kcal/mol each, as calculated from the f factor), the summarized relative contribution of these internucleotide phosphates is ΔG° approximately −4.1 ± 0.2 kcal/mol. A comparable value of ΔΔG° approximately −3.9 ± 0.2 kcal/mol may be calculated from the ratio of the Ki values for dTMP and d(pT)13–23 (average ΔG° = −4.0 ± 0.2 kcal/mol for n = 13–23). Thus, all contacts of OGG1 with 10–14 nt (including the dNMP unit with the higher affinity) of one strand of DNA provide ΔG° of −6.7 ± 0.3 kcal/mol at most. From the ratio of the average Ki values for ss and ds d(pN)13–23 including G11 (2.3- to 3.3-fold) characterizing the difference in the affinity of OGG1 for ds ODNs compared to ss ODNs, the contribution of the second strand to the affinity are only ΔG° approximately −0.5 … −0.7 kcal/mol. The X-ray structure of cognate and non-cognate OGG1:DNA complexes and stopped-flow data indicate that the enzyme distorts any DNA, creating a sharp kink, but fails to insert the non-damaged base into the active site pocket (21,22,25,27). Therefore, the active site of OGG1, as defined using the thermodynamic SILC approach, probably mostly interacts with the sugar-phosphate backbone of a dNMP unit within non-cognate DNA, which explains why the affinity of the enzyme for free dNMPs is greater than the affinity of its active site for dNMP units within long ODNs. Overall, the types of OGG1 interaction with non-cognate DNA can be summarized by the thermodynamic model shown in Figure 4.Figure 4.


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)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113562&req=5

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.
Mentions: Based on the measurements of binding affinity of OGG1 for various ligands, we have constructed a model summarizing the contribution of different interactions to DNA binding by OGG1. As mentioned above, extrapolation of the log(Ki) dependencies to n = 1 gives a Ki value of 12 mM, which characterizes the interaction of the active site of OGG1 with one of the nucleotides of d(pN)n bound to the protein. From this, the ΔG° of one non-specific dNMP interaction with the active site of OGG1 can be estimated as −2.6 kcal/mol. Since 12 phosphate groups of one strand of ds d(pN)13 interact with OGG1 through weak, additive contacts, (ΔG° approximately −0.34 kcal/mol each, as calculated from the f factor), the summarized relative contribution of these internucleotide phosphates is ΔG° approximately −4.1 ± 0.2 kcal/mol. A comparable value of ΔΔG° approximately −3.9 ± 0.2 kcal/mol may be calculated from the ratio of the Ki values for dTMP and d(pT)13–23 (average ΔG° = −4.0 ± 0.2 kcal/mol for n = 13–23). Thus, all contacts of OGG1 with 10–14 nt (including the dNMP unit with the higher affinity) of one strand of DNA provide ΔG° of −6.7 ± 0.3 kcal/mol at most. From the ratio of the average Ki values for ss and ds d(pN)13–23 including G11 (2.3- to 3.3-fold) characterizing the difference in the affinity of OGG1 for ds ODNs compared to ss ODNs, the contribution of the second strand to the affinity are only ΔG° approximately −0.5 … −0.7 kcal/mol. The X-ray structure of cognate and non-cognate OGG1:DNA complexes and stopped-flow data indicate that the enzyme distorts any DNA, creating a sharp kink, but fails to insert the non-damaged base into the active site pocket (21,22,25,27). Therefore, the active site of OGG1, as defined using the thermodynamic SILC approach, probably mostly interacts with the sugar-phosphate backbone of a dNMP unit within non-cognate DNA, which explains why the affinity of the enzyme for free dNMPs is greater than the affinity of its active site for dNMP units within long ODNs. Overall, the types of OGG1 interaction with non-cognate DNA can be summarized by the thermodynamic model shown in Figure 4.Figure 4.

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