Laboratory of Genomic and Protein Engineering

Dmitry O. Zharkov,
D.Biol.Sci., corr.member of RAS
Russian Science Support Foundation fellow (2004, 2005); Novosibirsk Region Government Young Scientist Award for Basic and Applied Research (2009); RFBR Award for Popular Science Writing (2009); member of the editorial board of “Journal of Biomolecular Structure and Dynamics”
Phone: +7(383)363-51-28; Email

• Fundamental mechanisms of DNA damage, DNA repair and cell response to genotoxic stress.
• Human diseases linked to genotoxic stress and DNA repair defects.
• Development of molecular biology tools employing DNA repair enzymes.
• Design of enzymes and multicomponent biomacromolecular systems with predicted functions and properties.
• Interaction of biomacromolecules and cells with artificial surfaces.

• X-ray crystallography and molecular dynamics were applied to clarify structures of several DNA repair proteins, including Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) covalently bound to DNA, E. coli endonuclease VIII E. coli (Nei), both free and covalently bound to DNA, bacteriophage T4 endonuclease V covalently bound to DNA, and free E. coli MutY DNA glycosylase. A comparison of these structures with other available structures of these enzymes revealed conformational changes in the enzymes and their DNA substrates occurring during the catalytic reaction.
• A complex of factors affecting the substrate specificity of Fpg and human 8-oxoguanine-DNA glycosylase (OGG1) has been studied. 8-Oxoguanine (oxoG) is an abundant DNA lesion that directs pre-mutagenic incorporation of dAMP during the replication. To avoid mutations, the repair of oxoG by Fpg in bacteria and OGG1 in humans must be efficient in oxoG:C pairs but not in oxoG:A mispairs. We have shown that under conditions close to those in the cell preference of Fpg and OGG1 for 8-oxoG:C but not for 8-oxoG:A is mainly due to high ionic strength and presence of Mg2+ ions. For OGG1, an additional contributor to the enzyme’s preferences is its specific stimulation by APEX1 endonuclease, an enzyme that catalyzes the next step of repair. We have also characterized polymorphic variants of OGG1 that differ from the wild-type protein in their activity and specificity.
• Human NEIL1 and NEIL2 proteins, homologs of bacterial Fpg and Nei, have been extensively studied. We found that these proteins possess 2’-deoxyribo-5’-phosphate lyase activity. NEIL1, together with OGG1, have been discovered to repair 8-oxoadenine lesions in DNA. NEIL1, OGG1, Fpg, and Nei turned out to be inhibited by Cd2+, Cu2+, and Zn2+ ions, an observation that may explain the co-mutagenic activity of these metals during oxidative stress. Parts of NEIL2 protein responsible for DNA binding have been identified.
• Search of specific sites in DNA can be performed using three main mechanisms: random three-dimensional diffusion (distributive search), random one-dimensional walk (processive search), and ATP-dependent directional one-dimensional translocation. An elementary event of one-dimensional walk may be a transfer of an enzyme from one position on DNA to an adjacent position without dissociation of the protein–DNA complex (sliding mechanism) or a dissociation of the protein molecule from the complex with DNA and immediate re-binding close to the previous site of binding (hopping mechanism). We have developed a new approach to quantitatively characterization of processive search and estimation of contribution of sliding and hopping mechanism to the search. This method has been used to analyseanalyse the mechanism of lesion search by E. coli uracil-DNA glycosylase, Fpg, and OGG1 proteins. It has been shown that these proteins use the processive search mode when the ionic strength is low and change to the distributive search when the ionic strength increases.
• The role of DNA repair in extension of runs of trinucleotide repeats and in the regulation of epigenetic methylation status has been investigated. The dependence of the repair efficiency on position of damage within a run of trinucleotide repeats have been discovered. MBD4 DNA glycosylase was shown to participate in active demethylation of CpG islands in DNA.

1. Joldybayeva B, Prorok P, Grin IR, Zharkov DO, Ishenko AA, Tudek B, Bissenbaev AK, Saparbaev M. Cloning and characterization of a wheat homologue of apurinic/apyrimidinic endonuclease Ape1L. PLoS One. 2014. 9(3):e92963.
2. Graifer D.M., Malygin A.A., Zharkov D.O., Karpova G.G. Eukaryotic ribosomal protein S3: A constituent of translational machinery and an extraribosomal player in various cellular processes. Biochimie. 2014. 99, 8-18.
3. Sattarova E.A., Sinitsyna O.I., Vasyunina E.A., Duzhak T.G., Kolosova N.G., Zharkov D.O., Nevinsky G.A. Age-dependent guanine oxidation in DNA of different brain regions of Wistar rats and prematurely aging OXYS rats. Biochim. Biophys. Acta - General Subjects. 2013. 1830(6), 3542-3552.
4. Kasymov R.D., Grin I.R., Endutkin A.V., Smirnov S.L., Ishchenko A.A., Saparbaev M.K., Zharkov D.O. Excision of 8-oxoguanine from methylated CpG dinucleotides by human 8-oxoguanine DNA glycosylase. FEBS Lett. 2013. 587(18), 3129-3134.
5. Prorok P., Alili D., Saint-Pierre C., Gasparutto D., Zharkov D.O., Ishchenko A.A., Tudek B., Saparbaev M.K. Uracil in duplex DNA is a substrate for the human nucleotide incision repair pathway. PNAS. 2013. 110(39), E3695-E3703
6. Lukina M.V., Popov A.V., Koval V.V., Vorobjev Y.N., Fedorova O.S., Zharkov D.O. DNA damage processing by human 8-oxoguanine-DNA glycosylase mutants with the occluded active site. J. Biol. Chem. 2013. 288(40), 28936-28947.
7. Popov A.V., Vorobjev Y.N., Zharkov D.O. MDTRA: A molecular dynamics trajectory analyzer with a graphical user interface. J. Comput. Chem. 2013. 34(4), 319-325.
8. Zharkov D.O. DNA oxidation. Encyclopedia of Biological Chemistry. Vol. 2. Lennarz W.J., Lane M.D. (Eds.) London – Burlington – San Diego: Academic Press. 2013, 77–81.
9. Popov A.V., Vorobjev Y.N., Zharkov D.O. MDTRA: A molecular dynamics trajectory analyzer with a graphical user interface. J. Comput. Chem. 2013. 34(4), 319–325.
10. Zharkov D.O. DNA oxidation. Encyclopedia of Biological Chemistry, Vol. 2. Lennarz W.J., Lane M.D. Eds. – London – Burlington – San Diego: Academic Press. 2013, 77–81.
11. Popov A.V., Vorobjev Yu.N., Zharkov D.O. MDTRA: A molecular dynamics trajectory analyser with a graphical user interface. J. Comput. Chem. 2013. 34(4), 319–325.
12. Morera S., Grin I.R., Vigouroux A., Couve S., Henriot V., Saparbaev M.K., Ishchenko A.A. Biochemical and structural characterization of the glycosylase domain of MBD4 bound to thymine and 5-hydroxymethyuracil-containing DNA. Nucleic Acids Res. 2012. 40(19), 9917–9926.
13. Kuznetsov N.A., Koval V.V., Zharkov D.O., Fedorova O.S. Conformational dynamics of interaction of Escherichia coli endonuclease VIII with DNA substrates. DNA Repair. 2012. 11(11), 884–891.
14. Pestryakov P.E., Zharkov D.O., Grin I.R., Fomina E.E., Kim E.R., Hamon L., Eliseeva I.A., Petruseva I.O., Curmi P.A., Ovchinnikov L.P., Lavrik O.I. Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1. J. Mol. Recognit. 2012. 25(4), 224–233.
15. Derevyanko A.G., Endutkin A.V., Ishchenko A.A., Saparbaev M.K., Zharkov D.O. Initiation of 8-oxoguanine base excision repair within trinucleotide tandem repeats. Biochemistry (Mosc.). 2012. 77(3), 270–279.
16. Kirpota O.O., Endutkin A.V., Ponomarenko M.P., Ponomarenko P.M., Zharkov D.O., Nevinsky G.A. Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase. Nucleic Acids Res. 2011. 39(11), 4836–4850.
17. Mechetin G.V., Zharkov D.O. Mechanism of translocation of uracil-DNA glycosylase from Escherichia coli between distributed lesions. Biochem. Biophys. Res. Commun. 2011. 414(2), 425–430.
18. Grin I.R., Zharkov D.O. Eukaryotic endonuclease VIII-like proteins: New components of the base excision DNA repair system. Biochemistry (Mosc.). 2011. 76(1), 80–93.
19. Mechetin G.V., Zharkov D.O. The mechanism of substrate search by base excision repair enzymes. Dokl. Biochem. Biophys. 2011. 437(1), 94–97.

RFBR (Grants of the Russian Foundation for Basic Research)

• N.13-04-01267 «Uracil-DNA glycosylases of poxviruses: biochemical characterization and evaluation as potential antiviral therapy targets» (2013-2014)
• N.14-04-01879 «Structural, dynamic and cellular factors governing the in vitro and in vivo substrate specificity of enzymes that repair oxidative DNA damage» (2014-2016)

Program of the Presidium of RAS N.6 “Molecular and Cell Biology” (2013–2015)

• N.12 “Mechanisms of regulation of cellular response to DNA and mononucleotide damage”

Russian President’ scolarship for young scientists and graduate students (2013–2015)

• SP-1716.2013.4 “Use of DNA repair enzymes for quality improvement of degraded DNA templates before PCR” (Dovgerd A.P.)