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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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Analysis of the inhibition type and estimation of Ki 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).
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Figure 2: Analysis of the inhibition type and estimation of Ki 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).

Mentions: All tested short cognate and non-cognate ss or ds ODNs inhibited the reaction of cleavage of oxoG catalyzed by OGG1. Using ds OG11 as a substrate, several other ODN substrates and inhibitors were shown to be competitive inhibitors towards it (Figure 2, Table 1). Therefore, Ki gives an estimate of binding affinity (Kd = Ki) of ODNs for the DNA-binding site of OGG1. In the absence of the inhibitor, the reaction was characterized by KM = 11 nM. Since most of the short ODNs had relatively low affinities for OGG1, the Ki values were calculated from the IC50 values using the equation corresponding to competitive inhibition, IC50 = 3Ki at S = 2KM (Table 1).Figure 2.


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)

Analysis of the inhibition type and estimation of Ki 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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Analysis of the inhibition type and estimation of Ki 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).
Mentions: All tested short cognate and non-cognate ss or ds ODNs inhibited the reaction of cleavage of oxoG catalyzed by OGG1. Using ds OG11 as a substrate, several other ODN substrates and inhibitors were shown to be competitive inhibitors towards it (Figure 2, Table 1). Therefore, Ki gives an estimate of binding affinity (Kd = Ki) of ODNs for the DNA-binding site of OGG1. In the absence of the inhibitor, the reaction was characterized by KM = 11 nM. Since most of the short ODNs had relatively low affinities for OGG1, the Ki values were calculated from the IC50 values using the equation corresponding to competitive inhibition, IC50 = 3Ki at S = 2KM (Table 1).Figure 2.

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