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The toxic action of different endogenous and exogenous agents leads to damage in genomic DNA. 8-Oxoguanine is one of the most often generated and highly mutagenic oxidative forms of damage in DNA. Normally, in human cells it is promptly removed by 8-oxoguanine-DNA-glycosylase hOGG1, the key DNA-repair enzyme. An association between the accumulation of oxidized guanine and an increased risk of harmful processes in organisms was already found. However, the detailed mechanism of damaged base recognition and removal is still unclear. To clarify the role of active site amino acids in the damaged base coordination and to reveal the elementary steps in the overall enzymatic process we investigated hOGG1 mutant forms with substituted amino acid residues in the enzyme base-binding pocket. Replacing the functional groups of the enzyme active site allowed us to change the rates of the individual steps of the enzymatic reaction. To gain further insight into the mechanism of hOGG1 catalysis a detailed pre-steady state kinetic study of this enzymatic process was carried out using the stopped-flow approach. The changes in the DNA structure after mixing with enzymes were followed by recording the FRET signal using Cy3/Cy5 labels in DNA substrates in the time range from milliseconds to hundreds of seconds. DNA duplexes containing non-damaged DNA, 8-oxoG, or an AP-site or its unreactive synthetic analogue were used as DNA-substrates. The kinetic parameters of DNA binding and damage processing were obtained for the mutant forms and for WT hOGG1. The analyses of fluorescence traces provided information about the DNA dynamics during damage recognition and removal. The kinetic study for the mutant forms revealed that all introduced substitutions reduced the efficiency of the hOGG1 activity; however, they played pivotal roles at certain elementary stages identified during the study. Taken together, our results gave the opportunity to restore the role of substituted amino acids and main "damaged base-amino acid" contacts, which provide an important link in the understanding the mechanism of the DNA repair process catalyzed by hOGG1.

The apurinic/apyrimidinic endonuclease from Saccharomyces cerevisiae Apn1 is one of the key enzymes involved in base excision repair of DNA lesions. A major function of the enzyme is to cleave the upstream phosphodiester bond of an apurinic/apyrimidinic site (AP-site), leading to the formation of a single-strand break with 3'-hydroxyl (OH) and 5'-deoxyribose phosphate (dRP) termini. In this study, the pre-steady-state kinetics and conformational dynamics of DNA substrates during their interaction with Apn1 were investigated. A stopped-flow method with detection of the fluorescence intensity of 2-aminopurine and pyrrolocytosine located adjacent or opposite to the damage was used. It was found that upon interaction with Apn1, both DNA strands undergo a number of rapid changes. The location of fluorescent analogs of heterocyclic bases in DNA does not influence the catalytic step of the reaction. Comparison of data obtained for yeast Apn1 and reported data (Kanazhevskaya, L. Yu., Koval, V. V., Vorobjev, Yu. N., and Fedorova, O. S. (2012) Biochemistry, 51, 1306-1321) for human Ape1 revealed some differences in their interaction with DNA substrates.

PELDOR (pulsed electron-electron double resonance) spectroscopy was applied to determine spin-spin distances in spin-labeled DNA duplexes (13-mer and 17-mer) containing the damaged sites 8-oxoguanine or uncleavable abasic site analogue tetrahydrofuran. The lesions were located in one strand of the DNA, and two nitroxyl spin labels were attached at the 5'- and 3'-ends of the complementary strand. PELDOR data allow us to obtain distances between the two spin labels in DNAs, which turned out to be around 5 nm for the 13-mer DNA and around 6 nm for 17-mer DNA. Results of PELDOR measurements were supported by molecular dynamics calculations. Study of the interaction of DNA fragments with DNA repair enzyme 8-oxoguanine-DNA glycosylase from E. coli (Fpg protein) showed that this interaction leads to a noticeable decrease of the distance between spin labels, which indicates the enzyme-induced bending of the DNA duplex. This bending may be important for the mechanisms of recognition of damaged sites by DNA repair enzymes.

Recent data on structural and biochemical features of human 8-oxoguanine DNA glycosylase (hOGG1) has enabled detailed evaluation of the mechanism by which the damaged DNA bases are recognized and eliminated from the chain. Pre-steady-state kinetic studies with recording of conformational transitions of the enzyme and DNA substrate significantly contribute to understanding of this mechanism. In this review we particularly focus on the interrelationship between the conformational changes of interacting molecules and kinetics of their interaction and on the nature of each elementary step during the enzymatic process. Exhaustive analysis of these data and detailed mechanism of hOGG1-catalyzed reaction are proposed.

Thermodynamics of interaction of phthalocyanine-oligonucleotide conjugates with single- and double-stranded DNA resulting in formation of duplexes and triplexes was measured by UV melting method. It was shown that a phthalocyanine moiety of conjugates stabilized the formation of duplexes and triplexes.

Photoactive derivatives of oligonucleotides are widely used as affinity reagents for the study of structures and functions of nucleic acids and proteins. Between them the binary reagents are the more attractive in the last time. They represent the tandem of two oligonucleotide derivatives complementary to a target sequence and carrying photoactive and sensitizing groups. The efficiency of target modification in this case depends on the mutual arrangement in the nick region of photoactive and sensitizing groups, attached to the oligonucleotides. The use of binary reagents in affinity modification permits to reach the high selectivity of the process. In this work we report our studies on the thermodynamic and structural peculiarities of complementary tandem complex between DNA target and binary oligonucleotide reagent. The complex consisted of the target d(TTGAAGGGGACCGC)and two 7-mer oligonucleotide conjugates,one of which was modified on its 3'-phosphate with a photoreactive p-azidote-trafluorobenzaldehydehydrazone-group,and the other one was linked through its 5'-phosphate to a sensitizing perylene-group. Optical melting curves and thermal changes in circular dichroism (CD)spectra were detected for all possible oligonucleotide and/or conjugate combinations.In addition,molecular modeling simulation of the complex structure was carried out. It was found that CD spectra did not show serious changes in the B-helix structure of the duplex. The interaction between perylene-and azido-groups at the oligonucleotide junction led to considerable increase in duplex stability. CD and molecular modeling data clearly indicated that perylene-group interacted with the duplex in an intercalative manner,but azido-group located on the side of DNA chain minor groove.

The synthesis of new metallophthalocyanine-oligonucleotide conjugates is reported. These conjugates can cause sequence-specific photosensitized or catalytic oxidation of DNA by molecular oxygen.

The cobalt(II) tetracarboxyphthalocyanine-deoxyribonucleotide pd(TCTTCCCA) conjugate was synthesized. The phthalocyanine N-succinimide ester prepared from phthalocyanine using DCC was mixed in DMF with an aqueous solution of the oligonucleotide bearing a 1,3-diaminopropane linker at the 5'-phosphate. The resulting conjugate was tested in the intraduplex reaction with target 14-mer and 22-mer oligonucleotides containing conjugate-complementary sequences. In the presence of O2 and a thiol (2-mercaptoethanol or DTT) as a coupled reducer or H2O2, sequence-specific DNA modification was observed that caused the cleavage of the target upon treatment with piperidine.

The kinetics of photomodification of oligodeoxyribonucleotide pd(GTGTGA) with a derivative of the complementary oligodeoxyribonucleotide pd(CACACA) bearing a 3-(n-azidotetrafluorobenzoylamino)propylamine residue (ArN3) at the terminal phosphate group was studied at 20 degrees C. It was found that the target's G3 residue is preferentially modified. Along with the transformation of the arylazide moiety, degradation of the oligonucleotide fragment of the reagent occurred with a partial loss of affinity. With the use of the reagent labeled at the 5'-end, sites of photomodification were found. From the dependence of the modification level on the reagent concentration at the initial time of irradiation, the association constant (Kx = (1.40 +/- 0.24) x 10(5) M-1) was determined. From the dependence of the modification level on the concentration of the pre-irradiated reagent, the constant of the transformed reagent-target association in solution (Kr = (2.49 +/- 0.30) x 10(4) M-1) was determined. From the time-dependence of the modification level [PZ]/P0, the rate constant for the limiting step of photomodification (k0 = (5.31 +/- 0.28) x 10(-4) S-1) and the modification efficiency of the target in the complex with the reagent (gamma = 1) were found.