Limits...
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.


Schematic representation of contacts in the complex between APE1 and DNAcontaining the F site (PDB ID 1DE8 [11]).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4837577&req=5

Figure 1: Schematic representation of contacts in the complex between APE1 and DNAcontaining the F site (PDB ID 1DE8 [11]).

Mentions: An analysis of the crystal structures of the free APE1 enzyme [8-10]and APE1-DNA covalent complexes [11-13] showed thatcatalysis in the APE1-DNA complex requires contacts whose formation leads toflipping of an AP site out of the doublehelix. Figure 1 presentsa scheme of the contacts in the enzymesubstrate complexbetween APE1 and DNA containing the F site lacking an OH-group in the C1’position of deoxyribose (PDB ID 1DE8). It is seen that enzyme amino acidresidues interact preferentially with one of the duplex strands to form usuallyhydrogen bonds and electrostatic contacts between DNA phosphate groups andamino acid side chains and also amide groups of peptide bonds of the protein.The enzyme active site is formed by Asp308, His309, Glu96, Asp210, Tyr171,Asn212, and Asn174 residues. The flipped out AP site conformation is stabilizedby Met270 and Arg177 residues. Met270 is embedded into the DNA minor groove,thereby displacing the base opposite to the AP site. The Arg177 residue isinserted on the DNA major groove side and forms a hydrogen bond with aphosphate group located downstream of the AP site. In the enzyme-substratecomplex, which is in a catalytically competent state, a phosphate residuelocated upstream of the AP site is coordinated by Asn174, Asn212, and His309residues. The catalytic reaction begins with the nucleophilic attack of a watermolecule that is coordinated, directly or indirectly through a Mg2+ion, by Asp210 on a 5’-phosphate group [11, 13].


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)

Schematic representation of contacts in the complex between APE1 and DNAcontaining the F site (PDB ID 1DE8 [11]).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematic representation of contacts in the complex between APE1 and DNAcontaining the F site (PDB ID 1DE8 [11]).
Mentions: An analysis of the crystal structures of the free APE1 enzyme [8-10]and APE1-DNA covalent complexes [11-13] showed thatcatalysis in the APE1-DNA complex requires contacts whose formation leads toflipping of an AP site out of the doublehelix. Figure 1 presentsa scheme of the contacts in the enzymesubstrate complexbetween APE1 and DNA containing the F site lacking an OH-group in the C1’position of deoxyribose (PDB ID 1DE8). It is seen that enzyme amino acidresidues interact preferentially with one of the duplex strands to form usuallyhydrogen bonds and electrostatic contacts between DNA phosphate groups andamino acid side chains and also amide groups of peptide bonds of the protein.The enzyme active site is formed by Asp308, His309, Glu96, Asp210, Tyr171,Asn212, and Asn174 residues. The flipped out AP site conformation is stabilizedby Met270 and Arg177 residues. Met270 is embedded into the DNA minor groove,thereby displacing the base opposite to the AP site. The Arg177 residue isinserted on the DNA major groove side and forms a hydrogen bond with aphosphate group located downstream of the AP site. In the enzyme-substratecomplex, which is in a catalytically competent state, a phosphate residuelocated upstream of the AP site is coordinated by Asn174, Asn212, and His309residues. The catalytic reaction begins with the nucleophilic attack of a watermolecule that is coordinated, directly or indirectly through a Mg2+ion, by Asp210 on a 5’-phosphate group [11, 13].

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.