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Thermodynamics of Damaged DNA Binding and Catalysis by Human AP Endonuclease 1.

Miroshnikova AD, Kuznetsova AA, Kuznetsov NA, Fedorova OS - Acta Naturae (2016 Jan-Mar)

Bottom Line: The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of "crystalline" water molecules from DNA grooves.The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5'-phosphate-2'-deoxyribose residue.It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences. Prosp. Acad. Lavrent'eva, 8, Novosibirsk, 630090, Russia;

ABSTRACT
Apurinic/apyrimidinic (AP) endonucleases play an important role in DNA repair and initiation of AP site elimination. One of the most topical problems in the field of DNA repair is to understand the mechanism of the enzymatic process involving the human enzyme APE1 that provides recognition of AP sites and efficient cleavage of the 5'-phosphodiester bond. In this study, a thermodynamic analysis of the interaction between APE1 and a DNA substrate containing a stable AP site analog lacking the C1' hydroxyl group (F site) was performed. Based on stopped-flow kinetic data at different temperatures, the steps of DNA binding, catalysis, and DNA product release were characterized. The changes in the standard Gibbs energy, enthalpy, and entropy of sequential specific steps of the repair process were determined. The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of "crystalline" water molecules from DNA grooves. The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5'-phosphate-2'-deoxyribose residue. It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step.

No MeSH data available.


Changes in the Trp fluorescence intensity during interaction between APE1 andthe F substrate at different temperatures. [APE1] = 1.0 μM, [F substrate]= 2.0 μM.
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Figure 4: Changes in the Trp fluorescence intensity during interaction between APE1 andthe F substrate at different temperatures. [APE1] = 1.0 μM, [F substrate]= 2.0 μM.

Mentions: To clarify the nature of the processes occurring during sequential stages of Fsite recognition in the DNA-substrate complex, catalysis, and enzyme-productcomplex dissociation, we conducted a stepwise thermodynamic analysis of theinteraction between APE1 and the F substrate. Stopped-flow measurements of theTrp fluorescence intensity provided kinetic curves characterizing theinteraction between APE1 and the 17- mer F substrate at one enzyme turnoverconditions and temperature of 10 to 37°C(Fig. 3). It isseen that the interaction between APE1 and the F substrate leads to multiphasechanges in the Trp fluorescence intensity. According to the previously obtaineddata [14, 15],a decrease in the fluorescence intensity in the initialpart of the kinetic curves characterizes the formation of a catalyticallycompetent complex. The catalytic reaction step leading to the formation ofproducts and subsequent dissociation of the enzyme-product complex isaccompanied by an increase in the Trp fluorescence intensity at longer times (> 1 s). As is evident from the kinetic curves(Fig. 4),both phases of the changes in the fluorescence intensity are temperature-dependent.


Thermodynamics of Damaged DNA Binding and Catalysis by Human AP Endonuclease 1.

Miroshnikova AD, Kuznetsova AA, Kuznetsov NA, Fedorova OS - Acta Naturae (2016 Jan-Mar)

Changes in the Trp fluorescence intensity during interaction between APE1 andthe F substrate at different temperatures. [APE1] = 1.0 μM, [F substrate]= 2.0 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837577&req=5

Figure 4: Changes in the Trp fluorescence intensity during interaction between APE1 andthe F substrate at different temperatures. [APE1] = 1.0 μM, [F substrate]= 2.0 μM.
Mentions: To clarify the nature of the processes occurring during sequential stages of Fsite recognition in the DNA-substrate complex, catalysis, and enzyme-productcomplex dissociation, we conducted a stepwise thermodynamic analysis of theinteraction between APE1 and the F substrate. Stopped-flow measurements of theTrp fluorescence intensity provided kinetic curves characterizing theinteraction between APE1 and the 17- mer F substrate at one enzyme turnoverconditions and temperature of 10 to 37°C(Fig. 3). It isseen that the interaction between APE1 and the F substrate leads to multiphasechanges in the Trp fluorescence intensity. According to the previously obtaineddata [14, 15],a decrease in the fluorescence intensity in the initialpart of the kinetic curves characterizes the formation of a catalyticallycompetent complex. The catalytic reaction step leading to the formation ofproducts and subsequent dissociation of the enzyme-product complex isaccompanied by an increase in the Trp fluorescence intensity at longer times (> 1 s). As is evident from the kinetic curves(Fig. 4),both phases of the changes in the fluorescence intensity are temperature-dependent.

Bottom Line: The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of "crystalline" water molecules from DNA grooves.The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5'-phosphate-2'-deoxyribose residue.It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences. Prosp. Acad. Lavrent'eva, 8, Novosibirsk, 630090, Russia;

ABSTRACT
Apurinic/apyrimidinic (AP) endonucleases play an important role in DNA repair and initiation of AP site elimination. One of the most topical problems in the field of DNA repair is to understand the mechanism of the enzymatic process involving the human enzyme APE1 that provides recognition of AP sites and efficient cleavage of the 5'-phosphodiester bond. In this study, a thermodynamic analysis of the interaction between APE1 and a DNA substrate containing a stable AP site analog lacking the C1' hydroxyl group (F site) was performed. Based on stopped-flow kinetic data at different temperatures, the steps of DNA binding, catalysis, and DNA product release were characterized. The changes in the standard Gibbs energy, enthalpy, and entropy of sequential specific steps of the repair process were determined. The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of "crystalline" water molecules from DNA grooves. The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5'-phosphate-2'-deoxyribose residue. It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step.

No MeSH data available.