Klenow fragment and DNA polymerase α-primase fromserva calf thymus in water-in-oil microemulsions

Formamidopyrimidine-DNA glycosylase (Fpg) excises oxidized purines from damaged DNA. The recent determination of the three-dimensional structure of the covalent complex of DNA with Escherichia coli Fpg, obtained by reducing the Schiff base intermediate formed during the reaction [Gilboa et al., J. Biol. Chem. 277 (2002) 19811] has revealed a number of potential specific and non-specific interactions between Fpg and DNA. We analyze the structural data for Fpg in the light of the kinetic and thermodynamic data obtained by the method of stepwise increase in ligand complexity to estimate relative contributions of individual nucleotide units of lesion-containing DNA to its total affinity for this enzyme [Ishchenko et al., Biochemistry 41 (2002) 7540]. Stopped-flow kinetic analysis that has allowed the dissection of Fpg catalysis in time [Fedorova et al., Biochemistry 41 (2002) 1520] is also correlated with the structural data.

Recognition of damaged DNA by Escherichia coli Fpg protein: insights from structural and kinetic data

X-ray analysis of enzyme–DNA interactions is very informative in revealing molecular contacts, but provides neither quantitative estimates of the relative importance of these contacts nor information on the relative contributions of specific and nonspecific interactions to the total affinity of enzymes for specific DNA. A stepwise increase in the ligand complexity approach is used to estimate the relative contributions of virtually every nucleotide unit of synthetic DNA containing abasic sites to its affinity for apurinic/apyrimidinic endonuclease (APE1) from human placenta. It was found that APE1 interacts with 9–10 nt units or base pairs of single-stranded and double-stranded ribooligonucleotides and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleotide phosphate groups. Such nonspecific interactions of APE1 with nearly every nucleotide within its DNA-binding cleft provides up to seven orders of magnitude (ΔG° ∼ −8.7 to −9.0 kcal/mol) of the enzyme affinity for any DNA substrate. In contrast, interactions with the abasic site together with other specific APE1–DNA interactions provide only one order of magnitude (ΔG° ∼ −1.1 to −1.5 kcal/mol) of the total affinity of APE1 for specific DNA. We conclude that the enzyme's specificity for abasic sites in DNA is mostly due to a great increase (six to seven orders of magnitude) in the reaction rate with specific DNA, with formation of the Michaelis complex contributing to the substrate preference only marginally.

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Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic/apyrimidinic endonuclease from human placenta

X-ray structural analysis provides no quantitative estimate of the relative contribution of specific and nonspecific or strong and weak interactions to the total affinity of enzymes for nucleic acids. We have shown that the interaction between enzymes and long nucleic acids at the molecular level can be successfully analyzed by the method of stepwise increase in ligand complexity (SILC). In the present review we summarize our studies of human uracil DNA glycosylase and apurinic/apyrimidinic endonuclease, E. coli 8-oxoguanine DNA glycosylase and RecA protein using the SILC approach. The relative contribution of structural (X-ray analysis data), thermodynamic, and catalytic factors to the discrimination of specific and nonspecific DNA by these enzymes at the stages of complex formation, the following changes in DNA and enzyme conformations and especially the catalysis of the reactions is discussed.

Structural, thermodynamic, and kinetic basis for the activities of some nucleic acid repair enzymes.

According to the currently accepted model, enzymes searching for specific recognition sequences or structural elements (modified nucleotides, breaks, single-stranded DNA fragments, etc.) slide at a high rate along DNA. Such sliding is possible only if the enzymes possess sufficiently high affinity for all DNA, sequence notwithstanding. Therefore, significant differences in their affinity for specific and nonspecific DNA sequences are unlikely, and the formation of a complex between an enzyme and its target DNA is not a basic factor of enzyme specificity. To elucidate such factors, we have analyzed many DNA replication, DNA repair, topoisomerization, integration, and recombination enzymes using a number of physicochemical methods, including the method of stepwise increase in ligand complexity developed in our laboratory. It has been shown that high affinity of all studied enzymes for long DNAs is provided by the formation of many weak contacts of the enzyme with all nucleotide units covered by the protein globule. The main role lies in the contact between positively charged amino acid residues and internucleoside phosphate groups; however, the contribution of each contact is very small, and the full contact interface usually resembles that characteristic of interactions between oppositely charged biopolymer surfaces. In some cases, a significant contribution to the affinity is made through hydrophobic and/or van der Waals interactions of the enzymes with nucleotide bases. On the whole, such nonspecific interactions provide for five to eight orders of enzyme affinity for DNA, depending on the enzyme. Specific interactions of enzymes with long DNAs, in contrast to their contacts with small ligands, are usually weak and comparable in efficiency with weak nonspecific contacts. The sum of specific interactions most often provides for approximately one or, rarely, two orders of affinity. According to structural data, DNA binding to any of the investigated enzymes is followed by a stage of DNA conformation adjustment, which includes partial or complete DNA melting, deformation of its backbone, stretching, compression, bending or kinking, eversion of nucleotides from the DNA helix, etc. The full set of such changes is specific for each individual enzyme. The fact that all enzyme-dependent changes in DNA are effected through weak specific (rather than strong) interactions is very important. Enzyme-specific changes in DNA conformation are required for effective adjustment of reacting orbitals to an accuracy of 10°–15°, which is possible only in the case of specific DNAs. A transition from nonspecific to specific DNA leads to an increase in the reaction rate (k
cat) by four to eight orders of magnitude. Thus, the stages of DNA conformation adjustment and catalysis proper provide for the high specificity of enzyme action.

The Role of Weak Specific and Nonspecific Interactions in Enzymatic Recognition and Conversion of Long DNAs

Pt2+ -containing derivatives of oligodeoxyribonucleotides were used to evaluate the ligand affinity to the template sites of Klenow fragment of DNA polymerase I from E. coli and DNA polymerase I fr...

Victor V. Khomov

Papers

The mechanism of recognition of templates by DNA polymerases from pro- and eukaryotes as revealed by affinity modification data.