Publications by authors named "Dmitry O Zharkov"

80 Publications

Molecular dynamics approach to identification of new OGG1 cancer-associated somatic variants with impaired activity.

J Biol Chem 2020 Dec 23. Epub 2020 Dec 23.

Laboratory of Genome and Protein Engineering, SB RAS Institute of Chemical Biology and Fundamental Medicine, Russian.

DNA of living cells is always exposed to damaging factors. To counteract the consequences of DNA lesions, cells have evolved several DNA repair systems, among which base excision repair is one of the most important. Many currently used antitumor drugs act by damaging DNA, and DNA repair often interferes with chemo- and radiotherapy in cancer cells. Tumors are usually extremely genetically heterogeneous, often bearing mutations in DNA repair genes. Thus, knowledge of the functionality of cancer-related variants of proteins involved in DNA damage response and repair is of great interest for personalization of cancer therapy. Although computational methods to predict the variant functionality have attracted much attention, at present they are mostly based on sequence conservation and make little use of modern capabilities in computational analysis of 3D protein structure. We have used molecular dynamics (MD) to model the structures of 20 clinically observed variants of a DNA repair enzyme, 8-oxoguanine-DNA glycosylase (OGG1). In parallel, we have experimentally characterized the activity, thermostability and DNA binding in a subset of these mutant proteins. Among the analyzed variants of OGG1, three (I145M, G202C, and V267M) were significantly functionally impaired and were successfully predicted by MD. Alone or in combination with sequence-based methods, MD may be an important functional prediction tool for cancer-related protein variants of unknown significance.
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http://dx.doi.org/10.1074/jbc.RA120.014455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948927PMC
December 2020

Displacement of Slow-Turnover DNA Glycosylases by Molecular Traffic on DNA.

Genes (Basel) 2020 07 30;11(8). Epub 2020 Jul 30.

Siberian Branch of the Russian Acasemy of Sciences Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., 630090 Novosibirsk, Russia.

In the base excision repair pathway, the initiating enzymes, DNA glycosylases, remove damaged bases and form long-living complexes with the abasic DNA product, but can be displaced by AP endonucleases. However, many nuclear proteins can move along DNA, either actively (such as DNA or RNA polymerases) or by passive one-dimensional diffusion. In most cases, it is not clear whether this movement is disturbed by other bound proteins or how collisions with moving proteins affect the bound proteins, including DNA glycosylases. We have used a two-substrate system to study the displacement of human OGG1 and NEIL1 DNA glycosylases by DNA polymerases in both elongation and diffusion mode and by D4, a passively diffusing subunit of a viral DNA polymerase. The OGG1-DNA product complex was disrupted by DNA polymerase β (POLβ) in both elongation and diffusion mode, Klenow fragment (KF) in the elongation mode and by D4. NEIL1, which has a shorter half-life on DNA, was displaced more efficiently. Hence, both possibly specific interactions with POLβ and nonspecific collisions (KF, D4) can displace DNA glycosylases from DNA. The protein movement along DNA was blocked by very tightly bound Cas9 RNA-targeted nuclease, providing an upper limit on the efficiency of obstacle clearance.
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http://dx.doi.org/10.3390/genes11080866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465369PMC
July 2020

Protein Engineering of DNA-Dependent Enzymes.

Adv Exp Med Biol 2020 ;1241:19-33

Novosibirsk State University, Novosibirsk, Russia.

Enzymes are extremely efficient natural catalysts of a variety of chemical reactions. Design of enzymes with new functions and properties has become one of the main goals of modern protein engineering. The field of protein engineering is growing intensively, and different strategies were developed for the creation of enzymes with new properties. While there is plenty of methods and instruments, all modern protein engineering strategies could be divided in two major groups, broadly based on the core ideas of rational design or directed evolution. DNA-dependent proteins present an important target of protein engineering due to their wide use in molecular cloning, bioanalytics, and genetic manipulations. Here we review examples of successful application of biochemical, structural and computational approaches belonging to both protein engineering strategies to create new proteins belonging to three important classes of DNA-dependent enzymes: CRISPR-associated nuclease Cas9, DNA polymerases, and DNA glycosylases. The review contains examples of successfully designed enzymes and discusses the most useful approaches in the engineering of these specific enzyme classes, problems restraining the development of this field, and future directions in the development and application of designed DNA-dependent enzymes.
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http://dx.doi.org/10.1007/978-3-030-41283-8_2DOI Listing
July 2020

Inhibitors of DNA Glycosylases as Prospective Drugs.

Int J Mol Sci 2020 Apr 28;21(9). Epub 2020 Apr 28.

SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., Novosibirsk 630090, Russia.

DNA glycosylases are enzymes that initiate the base excision repair pathway, a major biochemical process that protects the genomes of all living organisms from intrinsically and environmentally inflicted damage. Recently, base excision repair inhibition proved to be a viable strategy for the therapy of tumors that have lost alternative repair pathways, such as BRCA-deficient cancers sensitive to poly(ADP-ribose)polymerase inhibition. However, drugs targeting DNA glycosylases are still in development and so far have not advanced to clinical trials. In this review, we cover the attempts to validate DNA glycosylases as suitable targets for inhibition in the pharmacological treatment of cancer, neurodegenerative diseases, chronic inflammation, bacterial and viral infections. We discuss the glycosylase inhibitors described so far and survey the advances in the assays for DNA glycosylase reactions that may be used to screen pharmacological libraries for new active compounds.
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http://dx.doi.org/10.3390/ijms21093118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247160PMC
April 2020

Reading Targeted DNA Damage in the Active Demethylation Pathway: Role of Accessory Domains of Eukaryotic AP Endonucleases and Thymine-DNA Glycosylases.

J Mol Biol 2019 Dec 20. Epub 2019 Dec 20.

SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., Novosibirsk, 630090, Russia; Department of Natural Sciences, Novosibirsk State University, 2 Pirogova St., Novosibirsk, 630090, Russia. Electronic address:

Base excision DNA repair (BER) is an important process used by all living organisms to remove nonbulky lesions from DNA. BER is usually initiated by DNA glycosylases that excise a damaged base leaving an apurinic/apyrimidinic (AP) site, and an AP endonuclease then cuts DNA at the AP site, and the repair is completed by correct nucleotide insertion, end processing, and nick ligation. It has emerged recently that the BER machinery, in addition to genome protection, is crucial for active epigenetic demethylation in the vertebrates. This pathway is initiated by TET dioxygenases that oxidize the regulatory 5-methylcytosine, and the oxidation products are treated as substrates for BER. T:G mismatch-specific thymine-DNA glycosylase (TDG) and AP endonuclease 1 (APE1) catalyze the first two steps in BER-dependent active demethylation. In addition to the well-structured catalytic domains, these enzymes possess long tails that are structurally uncharacterized but involved in multiple interactions and regulatory functions. In this review, we describe the known roles of the tails in TDG and APE1, discuss the importance of order and disorder in their structure, and consider the evolutionary aspects of these accessory protein regions. We also propose that the tails may be important for the enzymes' oligomerization on DNA, an aspect of their function that only recently gained attention.
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http://dx.doi.org/10.1016/j.jmb.2019.12.020DOI Listing
December 2019

Requirements for DNA bubble structure for efficient cleavage by helix-two-turn-helix DNA glycosylases.

Mutagenesis 2020 02;35(1):119-128

Novosibirsk State University, Novosibirsk, Russia.

Oxidative DNA lesions, constantly generated by both endogenous and environmentally induced reactive oxygen species, are removed via the base excision repair pathway. In bacteria, Fpg and Nei DNA glycosylases, belonging to the helix-two-turn-helix (H2TH) structural superfamily, remove oxidised purines and pyrimidines, respectively. Interestingly, the human H2TH family glycosylases, NEIL1, NEIL2 and NEIL3, have been reported to prefer oxidative lesions in DNA bubbles or single-stranded DNA. It had been hypothesised that NEIL2 might be involved in the repair of lesions in transcription bubbles; however, bubble-like structures may appear in other cellular contexts such as displacement loops (D-loops) associated with transcription, recombination or telomere maintenance. The activities of bacterial Fpg and Nei on bubble substrates were not addressed. Also, it is not known whether H2TH enzymes process bubbles containing the third DNA or RNA strand, and how the bubble length and position of the lesion within a bubble affect the excision. We have investigated the removal of 8-oxoguanine (8-oxoG) and 5,6-dihydrouracil (DHU) by Escherichia coli Fpg and Nei and human NEIL1 and NEIL2 from single-strand oligonucleotides, perfect duplexes, bubbles with different numbers of unpaired bases (6-30), bubbles containing the lesion in different positions and D-loops with the third strand made of DNA or RNA. Fpg, NEIL1 and NEIL2 efficiently excised lesions located within bubbles, with NEIL1 and NEIL2 being specific for DHU, and Fpg removing both 8-oxoG and DHU. Nei, in contrast, was significantly active only on DHU located in double-stranded DNA. Fpg and NEIL1 also tolerated the presence of the third strand of either DNA or RNA in D-loops if the lesion was in the single-stranded part, and Fpg, Nei and NEIL1 excised lesions from the double-stranded DNA part of D-loops. The presence of an additional unpaired 5'-tail of DNA or RNA did not affect the activity. No significant position preference for lesions in a 12-mer bubble was found. Overall, the activities of Fpg, NEIL1 and NEIL2 on these non-canonical substrates are consistent with the possibility that these enzymes may participate in the repair in structures arising during transcription or homologous recombination.
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http://dx.doi.org/10.1093/mutage/gez047DOI Listing
February 2020

Mechanistic insight into the role of Poly(ADP-ribosyl)ation in DNA topology modulation and response to DNA damage.

Mutagenesis 2020 02;35(1):107-118

Groupe «Réparation de l'ADN», Equipe Labellisée par la Ligue Nationale Contre le Cancer, Université Paris-Sud, Gustave Roussy Cancer Campus, Villejuif Cedex, France.

Genotoxic stress generates single- and double-strand DNA breaks either through direct damage by reactive oxygen species or as intermediates of DNA repair. Failure to detect and repair DNA strand breaks leads to deleterious consequences such as chromosomal aberrations, genomic instability and cell death. DNA strand breaks disrupt the superhelical state of cellular DNA, which further disturbs the chromatin architecture and gene activity regulation. Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyse the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are regarded as DNA damage sensors that, upon activation by strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. Noteworthy, the regularly branched structure of poly(ADP-ribose) polymer suggests that the mechanism of its synthesis may involve circular movement of PARP1 around the DNA helix, with a branching point in PAR corresponding to one complete 360° turn. We propose that PARP1 stays bound to a DNA strand break end, but rotates around the helix displaced by the growing poly(ADP-ribose) chain, and that this rotation could introduce positive supercoils into damaged chromosomal DNA. This topology modulation would enable nucleosome displacement and chromatin decondensation around the lesion site, facilitating the access of DNA repair proteins or transcription factors. PARP1-mediated DNA supercoiling can be transmitted over long distances, resulting in changes in the high-order chromatin structures. The available structures of PARP1 are consistent with the strand break-induced PAR synthesis as a driving force for PARP1 rotation around the DNA axis.
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http://dx.doi.org/10.1093/mutage/gez045DOI Listing
February 2020

Mechanism of stimulation of DNA binding of the transcription factors by human apurinic/apyrimidinic endonuclease 1, APE1.

DNA Repair (Amst) 2019 10 31;82:102698. Epub 2019 Aug 31.

Groupe «Réparation de l'ADN», Equipe Labellisée par la Ligue Nationale Contre le Cancer, CNRS UMR8200, Université Paris-Sud, Université Paris-Saclay, Gustave Roussy Cancer Campus, F-94805 Villejuif Cedex, France. Electronic address:

Aerobic respiration generates reactive oxygen species (ROS), which can damage nucleic acids, proteins and lipids. A number of transcription factors (TFs) contain redox-sensitive cysteine residues at their DNA-binding sites, hence ROS-induced thiol oxidation strongly inhibits their recognition of the cognate DNA sequences. Major human apurinic/apyrimidinic (AP) endonuclease 1 (APE1/APEX1/HAP-1), referred also as a redox factor 1 (Ref-1), stimulates the DNA binding activities of the oxidized TFs such as AP-1 and NF-κB. Also, APE1 participates in the base excision repair (BER) and nucleotide incision repair (NIR) pathways to remove oxidative DNA base damage. At present, the molecular mechanism underlying the TF-stimulating/redox function of APE1 and its biological role remains disputed. Here, we provide evidence that, instead of direct cysteine reduction in TFs by APE1, APE1-catalyzed NIR and TF-stimulating activities may be based on transient cooperative binding of APE1 to DNA and induction of conformational changes in the helix. The structure of DNA duplex strongly influences NIR and TF-stimulating activities. Homologous plant AP endonucleases lacking conserved cysteine residues stimulate DNA binding of the p50 subunit of NF-κB. APE1 acts synergistically with low-molecular-weight reducing agents on TFs. Finally, APE1 stimulates DNA binding of the redox-insensitive p50-C62S mutant protein. Electron microscopy imaging of APE1 complexes with DNA revealed preferential polymerization of APE1 on the gapped and intrinsically curved DNA duplexes. Molecular modeling offers a structural explanation how full-length APE1 can oligomerize on DNA. In conclusion, we propose that DNA-directed APE1 oligomerization can be regarded as a substitute for diffusion of APE1 along the DNA contour to probe for anisotropic flexibility. APE1 oligomers exacerbate pre-existing distortions in DNA and enable both NIR activity and DNA binding by TFs regardless of their oxidation state.
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http://dx.doi.org/10.1016/j.dnarep.2019.102698DOI Listing
October 2019

Isoforms of Base Excision Repair Enzymes Produced by Alternative Splicing.

Int J Mol Sci 2019 Jul 3;20(13). Epub 2019 Jul 3.

Novosibirsk State University, 1 Pirogova St., 630090 Novosibirsk, Russia.

Transcripts of many enzymes involved in base excision repair (BER) undergo extensive alternative splicing, but functions of the corresponding alternative splice variants remain largely unexplored. In this review, we cover the studies describing the common alternatively spliced isoforms and disease-associated variants of DNA glycosylases, AP-endonuclease 1, and DNA polymerase beta. We also discuss the roles of alternative splicing in the regulation of their expression, catalytic activities, and intracellular transport.
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http://dx.doi.org/10.3390/ijms20133279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6651865PMC
July 2019

Base Excision DNA Repair Deficient Cells: From Disease Models to Genotoxicity Sensors.

Curr Pharm Des 2019 ;25(3):298-312

Novosibirsk State University, 2 Pirogova St., Novosibirsk 630090, Russian Federation.

Base excision DNA repair (BER) is a vitally important pathway that protects the cell genome from many kinds of DNA damage, including oxidation, deamination, and hydrolysis. It involves several tightly coordinated steps, starting from damaged base excision and followed by nicking one DNA strand, incorporating an undamaged nucleotide, and DNA ligation. Deficiencies in BER are often embryonic lethal or cause morbid diseases such as cancer, neurodegeneration, or severe immune pathologies. Starting from the early 1980s, when the first mammalian cell lines lacking BER were produced by spontaneous mutagenesis, such lines have become a treasure trove of valuable information about the mechanisms of BER, often revealing unexpected connections with other cellular processes, such as antibody maturation or epigenetic demethylation. In addition, these cell lines have found an increasing use in genotoxicity testing, where they provide increased sensitivity and representativity to cell-based assay panels. In this review, we outline current knowledge about BER-deficient cell lines and their use.
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http://dx.doi.org/10.2174/1381612825666190319112930DOI Listing
February 2020

Critical Sites of DNA Backbone Integrity for Damaged Base Removal by Formamidopyrimidine-DNA Glycosylase.

Biochemistry 2019 06 3;58(24):2740-2749. Epub 2019 Jun 3.

SB RAS Institute of Chemical Biology and Fundamental Medicine , 8 Lavrentieva Avenue , Novosibirsk 630090 , Russia.

DNA glycosylases, the enzymes that initiate base excision DNA repair, recognize damaged bases through a series of precisely orchestrated movements. Most glycosylases sharply kink the DNA axis at the lesion site and extrude the target base from the DNA double helix into the enzyme's active site. Little attention has been paid so far to the role of the physical continuity of the DNA backbone in allowing the required conformational distortion. Here, we analyze base excision by formamidopyrimidine-DNA glycosylase (Fpg) from substrates keeping all phosphates but containing a nick within three nucleotides of the lesion in either DNA strand. Four phosphoester linkages at the damaged nucleotide and two nucleotides 3' to it were essential for Fpg activity, while the breakage of the others, even at the same critical phosphates, had no effect or even stimulated the reaction. Reduction of the likelihood of hydrogen bonding at the nicks by using dideoxynucleotides as their 3'-terminal groups was more detrimental for the activity. All phosphoester bonds in the complementary strand were dispensable for base excision, but nicks close to the orphaned nucleotide caused early termination of damaged strand cleavage. Elastic network analysis of Fpg-DNA structures showed that the vibrational motions of the critical phosphates are strongly correlated, in part due to the presence of the protein. Overall, our results suggest that mechanical forces propagating along the DNA backbone play a critical role in the correct conformational distortion of DNA by Fpg and possibly by other target base-everting DNA glycosylases.
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http://dx.doi.org/10.1021/acs.biochem.9b00134DOI Listing
June 2019

Conformational Dynamics of Damage Processing by Human DNA Glycosylase NEIL1.

J Mol Biol 2019 03 1;431(6):1098-1112. Epub 2019 Feb 1.

SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., Novosibirsk 630090, Russia; Novosibirsk State University, 2 Pirogova St., Novosibirsk 630090, Russia. Electronic address:

Endonuclease VIII-like protein 1 (NEIL1) is a DNA repair enzyme found in higher eukaryotes, including humans. It belongs to the helix-two turn-helix (H2TH) structural superfamily together with Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei), and removes a variety of oxidized purine and pyrimidine bases from DNA. Structural, modeling and kinetic studies have established that the bacterial H2TH superfamily enzymes proceed through several conformational intermediates while recognizing and removing their cognate lesions. Here we apply stopped-flow kinetics with detection of intrinsic Trp fluorescence and Förster resonance energy transfer fluorescence to follow the conformational dynamics of human NEIL1 and DNA when the enzyme interacts with undamaged DNA, or DNA containing cleavable or non-cleavable abasic sites, or dihydrouracil lesions. NEIL1 processed a natural abasic site and a damaged base in DNA equally well but showed an additional fluorescently discernible step when DHU was present, likely reflecting additional rearrangements during base eversion into the enzyme's active site. With undamaged DNA and DNA containing a non-cleavable abasic site analog, (3-hydroxytetrahydrofuran-2-yl)methyl phosphate, NEIL1 was diverted to a non-productive DNA conformation early in the reaction. Our results support the view of NEIL1 as an enzyme that actively destabilizes damaged DNA and uses multiple checkpoints along the reaction coordinate to drive substrate lesions into the active site while rejecting normal bases and non-substrate lesions.
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http://dx.doi.org/10.1016/j.jmb.2019.01.030DOI Listing
March 2019

Aberrant repair initiated by the adenine-DNA glycosylase does not play a role in UV-induced mutagenesis in .

PeerJ 2018 5;6:e6029. Epub 2018 Dec 5.

Groupe «Réparation de l'ADN», Equipe Labellisée par la Ligue Nationale Contre le Cancer, CNRS UMR8200, Université Paris-Sud, Gustave Roussy Cancer Campus, Villejuif, France.

Background: DNA repair is essential to counteract damage to DNA induced by endo- and exogenous factors, to maintain genome stability. However, challenges to the faithful discrimination between damaged and non-damaged DNA strands do exist, such as mismatched pairs between two regular bases resulting from spontaneous deamination of 5-methylcytosine or DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved the mismatch-specific DNA glycosylases that can recognize and remove regular DNA bases in the mismatched DNA duplexes. The adenine-DNA glycosylase (MutY/MicA) protects cells against oxidative stress-induced mutagenesis by removing adenine which is mispaired with 7,8-dihydro-8-oxoguanine (8oxoG) in the base excision repair pathway. However, MutY does not discriminate between template and newly synthesized DNA strands. Therefore the ability to remove A from 8oxoG•A mispair, which is generated via misincorporation of an 8-oxo-2'-deoxyguanosine-5'-triphosphate precursor during DNA replication and in which A is the template base, can induce A•T→C•G transversions. Furthermore, it has been demonstrated that human MUTYH, homologous to the bacterial MutY, might be involved in the aberrant processing of ultraviolet (UV) induced DNA damage.

Methods: Here, we investigated the role of MutY in UV-induced mutagenesis in . MutY was probed on DNA duplexes containing cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproduct (6-4PP). UV irradiation of induces Save Our Souls (SOS) response characterized by increased production of DNA repair enzymes and mutagenesis. To study the role of MutY in vivo, the mutation frequencies to rifampicin-resistant (Rif) after UV irradiation of wild type and mutant strains were measured.

Results: We demonstrated that MutY does not excise Adenine when it is paired with CPD and 6-4PP adducts in duplex DNA. At the same time, MutY excises Adenine in A•G and A•8oxoG mispairs. Interestingly, strains, which have elevated spontaneous mutation rate, exhibited low mutational induction after UV exposure as compared to MutY-proficient strains. However, sequence analysis of Rif mutants revealed that the frequencies of C→T transitions dramatically increased after UV irradiation in both MutY-proficient and -deficient strains.

Discussion: These findings indicate that the bacterial MutY is not involved in the aberrant DNA repair of UV-induced DNA damage.
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http://dx.doi.org/10.7717/peerj.6029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286661PMC
December 2018

Transient protein-protein complexes in base excision repair.

J Biomol Struct Dyn 2019 10 31;37(17):4407-4418. Epub 2018 Dec 31.

SB RAS Institute of Chemical Biology and Fundamental Medicine , Novosibirsk , Russia.

Transient protein-protein complexes are of great importance for organizing multiple enzymatic reactions into productive reaction pathways. Base excision repair (BER), a process of critical importance for maintaining genome stability against a plethora of DNA-damaging factors, involves several enzymes, including DNA glycosylases, AP endonucleases, DNA polymerases, DNA ligases and accessory proteins acting sequentially on the same damaged site in DNA. Rather than being assembled into one stable multisubunit complex, these enzymes pass the repair intermediates between them in a highly coordinated manner. In this review, we discuss the nature and the role of transient complexes arising during BER as deduced from structural and kinetic data. Almost all of the transient complexes are DNA-mediated, although some may also exist in solution and strengthen under specific conditions. The best-studied example, the interactions between DNA glycosylases and AP endonucleases, is discussed in more detail to provide a framework for distinguishing between stable and transient complexes based on the kinetic data. Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2018.1553741DOI Listing
October 2019

Oxidative damage to epigenetically methylated sites affects DNA stability, dynamics and enzymatic demethylation.

Nucleic Acids Res 2018 11;46(20):10827-10839

Chemistry Department, Western Washington University, 516 High St., Bellingham, WA 98225-9150, USA.

DNA damage can affect various regulatory elements of the genome, with the consequences for DNA structure, dynamics, and interaction with proteins remaining largely unexplored. We used solution NMR spectroscopy, restrained and free molecular dynamics to obtain the structures and investigate dominant motions for a set of DNA duplexes containing CpG sites permuted with combinations of 5-methylcytosine (mC), the primary epigenetic base, and 8-oxoguanine (oxoG), an abundant DNA lesion. Guanine oxidation significantly changed the motion in both hemimethylated and fully methylated DNA, increased base pair breathing, induced BI→BII transition in the backbone 3' to the oxoG and reduced the variability of shift and tilt helical parameters. UV melting experiments corroborated the NMR and molecular dynamics results, showing significant destabilization of all methylated contexts by oxoG. Notably, some dynamic and thermodynamic effects were not additive in the fully methylated oxidized CpG, indicating that the introduced modifications interact with each other. Finally, we show that the presence of oxoG biases the recognition of methylated CpG dinucleotides by ROS1, a plant enzyme involved in epigenetic DNA demethylation, in favor of the oxidized DNA strand. Thus, the conformational and dynamic effects of spurious DNA oxidation in the regulatory CpG dinucleotide can have far-reaching biological consequences.
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http://dx.doi.org/10.1093/nar/gky893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237784PMC
November 2018

Characterization of biochemical properties of an apurinic/apyrimidinic endonuclease from Helicobacter pylori.

PLoS One 2018 15;13(8):e0202232. Epub 2018 Aug 15.

National Center for Biotechnology, Astana, Kazakhstan.

Apurinic/apyrimidinic (AP) endonucleases play critical roles in the repair of abasic sites and strand breaks in DNA. Complete genome sequences of Helicobacter pylori reveal that this bacterial specie has a single AP endonuclease. An H. pylori homolog of Xth (HpXth) is a member of exonuclease III family, which is represented by Escherichia coli Xth. Currently, it remains unknown whether this single AP endonuclease has DNA repair activities similar to those of its counterpart in E. coli and other bacteria. We report that HpXth possesses efficient AP site cleavage, 3'-repair phosphodiesterase, and 3'-phosphatase activities but not the nucleotide incision repair function. Optimal reaction conditions for HpXth's AP endonuclease activity are low ionic strength, high Mg2+ concentration, pH in the range 7-8, and temperature 30 °C. The kinetic parameters measured under steady-state conditions showed that HpXth removes the AP site, 3'-blocking sugar-phosphate, and 3'-terminal phosphate in DNA strand breaks with good efficiency (kcat/KM = 1240, 44, and 5,4 μM-1·min-1, respectively), similar to that of E. coli Xth. As expected, the presence of HpXth protein in AP endonuclease-deficient E. coli xth nfo strain significantly reduced the sensitivity to an alkylating agent and H2O2. Mutation of active site residue D144 in HpXth predicted to be essential for catalysis resulted in a complete loss of enzyme activities. Several important structural features of HpXth were uncovered by homology modeling and phylogenetic analysis. Our data show the DNA substrate specificity of H. pylori AP endonuclease and suggest that HpXth counteracts the genotoxic effects of DNA damage generated by endogenous and host-imposed factors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202232PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093668PMC
February 2019

Residue coevolution reveals functionally important intramolecular interactions in formamidopyrimidine-DNA glycosylase.

DNA Repair (Amst) 2018 09 19;69:24-33. Epub 2018 Jul 19.

SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., Novosibirsk 630090, Russia; Novosibirsk State University, 2 Pirogova St., Novosibirsk 630090, Russia. Electronic address:

In protein evolution, functionally important intramolecular interactions, such as polar bridges or hydrophobic interfaces, tend to be conserved. We have analyzed coevolution of physicochemical properties in pairs of amino acid residues in the formamidopyrimidine-DNA glycosylase (Fpg) protein family, identified three conserved polar bridges (Arg54-Glu131, Gln234-Arg244, and Tyr170-Ser208 in the E. coli protein) located in known functional regions of the protein, and analyzed their roles by site-directed mutagenesis. The structure and molecular dynamic modeling showed that the coevolving pairs do not form isolated bridges but rather participate in tight local clusters of hydrogen bonds. The Arg54-Glu131 bridge, connecting the N- and C-terminal domains, was important for DNA binding, as its abolishment or even ion pair reversal inactivated Fpg and greatly decreased the enzyme's affinity for DNA. Mutations of the Gln234-Arg244 bridge, located at the base of the single Fpg β-hairpin zinc finger, did not affect the activity but sharply decreased the melting temperature of the protein, with the bridge reversal partially restoring the thermal stability. Finally, Tyr170 mutation to Phe decreased Fpg binding but did not fully inactivate the protein, whereas Ser208 replacement with Ala had no effect; molecular dynamics showed that in both wild-type and S208 A Fpg, Tyr170 quickly re-orients to form an alternative set of hydrogen bonds. Thus, the coevolution analysis approach, combined with biochemical and computational studies, provides a powerful tool for understanding intramolecular interactions important for the function of DNA repair enzymes.
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http://dx.doi.org/10.1016/j.dnarep.2018.07.004DOI Listing
September 2018

Variable termination sites of DNA polymerases encountering a DNA-protein cross-link.

PLoS One 2018 1;13(6):e0198480. Epub 2018 Jun 1.

Laboratory of Genome and Protein Engineering, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.

DNA-protein cross-links (DPCs) are important DNA lesions induced by endogenous crosslinking agents such as formaldehyde or acetaldehyde, as well as ionizing radiation, cancer chemotherapeutic drugs, and abortive action of some enzymes. Due to their very bulky nature, they are expected to interfere with DNA and RNA synthesis and DNA repair. DPCs are highly genotoxic and the ability of cells to deal with them is relevant for many chemotherapeutic interventions. However, interactions of DNA polymerases with DPCs have been poorly studied due to the lack of a convenient experimental model. We have used NaBH4-induced trapping of E. coli formamidopyrimidine-DNA glycosylase with DNA to construct model DNA polymerase substrates containing a DPC in single-stranded template, or in the template strand of double-stranded DNA, or in the non-template (displaced) strand of double-stranded DNA. Nine DNA polymerases belonging to families A, B, X, and Y were studied with respect to their behavior upon encountering a DPC: Klenow fragment of E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, Pyrococcus furiosus DNA polymerase, Sulfolobus solfataricus DNA polymerase IV, human DNA polymerases β, κ and λ, and DNA polymerases from bacteriophages T4 and RB69. Although none were able to fully bypass DPCs in any context, Family B DNA polymerases (T4, RB69) and Family Y DNA polymerase IV were able to elongate the primer up to the site of the cross-link if a DPC was located in single-stranded template or in the displaced strand. In other cases, DNA synthesis stopped 4-5 nucleotides before the site of the cross-link in single-stranded template or in double-stranded DNA if the polymerases could displace the downstream strand. We suggest that termination of DNA polymerases on a DPC is mostly due to the unrelieved conformational strain experienced by the enzyme when pressing against the cross-linked protein molecule.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198480PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983568PMC
December 2018

Molecular dynamics simulation of the opposite-base preference and interactions in the active site of formamidopyrimidine-DNA glycosylase.

BMC Struct Biol 2017 05 8;17(1). Epub 2017 May 8.

SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., Novosibirsk, 630090, Russia.

Background: Formamidopyrimidine-DNA glycosylase (Fpg) removes abundant pre-mutagenic 8-oxoguanine (oxoG) bases from DNA through nucleophilic attack of its N-terminal proline at C1' of the damaged nucleotide. Since oxoG efficiently pairs with both C and A, Fpg must excise oxoG from pairs with C but not with A, otherwise a mutation occurs. The crystal structures of several Fpg-DNA complexes have been solved, yet no structure with A opposite the lesion is available.

Results: Here we use molecular dynamic simulation to model interactions in the pre-catalytic complex of Lactococcus lactis Fpg with DNA containing oxoG opposite C or A, the latter in either syn or anti conformation. The catalytic dyad, Pro1-Glu2, was modeled in all four possible protonation states. Only one transition was observed in the experimental reaction rate pH dependence plots, and Glu2 kept the same set of interactions regardless of its protonation state, suggesting that it does not limit the reaction rate. The adenine base opposite oxoG was highly distorting for the adjacent nucleotides: in the more stable syn models it formed non-canonical bonds with out-of-register nucleotides in both the damaged and the complementary strand, whereas in the anti models the adenine either formed non-canonical bonds or was expelled into the major groove. The side chains of Arg109 and Phe111 that Fpg inserts into DNA to maintain its kinked conformation tended to withdraw from their positions if A was opposite to the lesion. The region showing the largest differences in the dynamics between oxoG:C and oxoG:A substrates was unexpectedly remote from the active site, located near the linker joining the two domains of Fpg. This region was also highly conserved among 124 analyzed Fpg sequences. Three sites trapping water molecules through multiple bonds were identified on the protein-DNA interface, apparently helping to maintain enzyme-induced DNA distortion and participating in oxoG recognition.

Conclusion: Overall, the discrimination against A opposite to the lesion seems to be due to incorrect DNA distortion around the lesion-containing base pair and, possibly, to gross movement of protein domains connected by the linker.
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http://dx.doi.org/10.1186/s12900-017-0075-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5422863PMC
May 2017

Recognition but no repair of abasic site in single-stranded DNA by human ribosomal uS3 protein residing within intact 40S subunit.

Nucleic Acids Res 2017 04;45(7):3833-3843

Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 630090, Russia.

Isolated human ribosomal protein uS3 has extra-ribosomal functions including those related to base excision DNA repair, e.g. AP lyase activity that nicks double-stranded (ds) DNA 3΄ to the abasic (AP) site. However, the ability of uS3 residing within ribosome to recognize and cleave damaged DNA has never been addressed. Here, we compare interactions of single-stranded (ss) DNA and dsDNA bearing AP site with human ribosome-bound uS3 and with the isolated protein, whose interactions with ssDNA were not yet studied. The AP lyase activity of free uS3 was much higher with ssDNA than with dsDNA, whereas ribosome-bound uS3 was completely deprived of this activity. Nevertheless, an exposed peptide of ribosome-bound uS3 located far away from the putative catalytic center previously suggested for isolated uS3 cross-linked to full-length uncleaved ssDNA, but not to dsDNA. In contrast, free uS3 cross-linked mainly to the 5΄-part of the damaged DNA strand after its cleavage at the AP site. ChIP-seq analysis showed preferential uS3 binding to nucleolus-associated chromatin domains. We conclude that free and ribosome-bound uS3 proteins interact with AP sites differently, exhibiting their non-translational functions in DNA repair in and around the nucleolus and in regulation of DNA damage response in looped DNA structures, respectively.
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http://dx.doi.org/10.1093/nar/gkx052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397187PMC
April 2017

DNA Deformation-Coupled Recognition of 8-Oxoguanine: Conformational Kinetic Gating in Human DNA Glycosylase.

J Am Chem Soc 2017 02 8;139(7):2682-2692. Epub 2017 Feb 8.

Novosibirsk State University , 2 Pirogova Street, Novosibirsk 630090, Russia.

8-Oxoguanine (8-oxoG), a mutagenic DNA lesion generated under oxidative stress, differs from its precursor guanine by only two substitutions (O and H). Human 8-oxoguanine glycosylase 1 (OGG1) can locate and remove 8-oxoG through extrusion and excision. To date, it remains unclear how OGG1 efficiently distinguishes 8-oxoG from a large excess of undamaged DNA bases. We recently showed that formamidopyrimidine-DNA glycosylase (Fpg), a bacterial functional analog of OGG1, can selectively facilitate eversion of oxoG by stabilizing several intermediate states, and it is intriguing whether OGG1 also employs a similar mechanism in lesion recognition. Here, we use molecular dynamics simulations to explore the mechanism by which OGG1 discriminates between 8-oxoG and guanine along the base-eversion pathway. The MD results suggest an important role for kinking of the DNA by the glycosylase, which positions DNA phosphates in a way that assists lesion recognition during base eversion. The computational predictions were validated through experimental enzyme assays on phosphorothioate substrate analogs. Our simulations suggest that OGG1 distinguishes between 8-oxoG and G using their chemical dissimilarities not only at the active site but also at earlier stages during base eversion, and this mechanism is at least partially conserved in Fpg despite a lack of structural homology. The similarity also suggests that lesion recognition through multiple gating steps may be a common theme in DNA repair. Our results provide new insight into how enzymes can exploit kinetics and DNA conformational changes to probe the chemical modifications present in DNA lesions.
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http://dx.doi.org/10.1021/jacs.6b11433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5321822PMC
February 2017

Aberrant base excision repair pathway of oxidatively damaged DNA: Implications for degenerative diseases.

Free Radic Biol Med 2017 06 24;107:266-277. Epub 2016 Nov 24.

Groupe «Réparation de l'ADN», Equipe Labellisée par la Ligue Nationale Contre le Cancer, CNRS UMR8200, Université Paris-Sud, Gustave Roussy Cancer Campus, F-94805 Villejuif Cedex, France. Electronic address:

In cellular organisms composition of DNA is constrained to only four nucleobases A, G, T and C, except for minor DNA base modifications such as methylation which serves for defence against foreign DNA or gene expression regulation. Interestingly, this severe evolutionary constraint among other things demands DNA repair systems to discriminate between regular and modified bases. DNA glycosylases specifically recognize and excise damaged bases among vast majority of regular bases in the base excision repair (BER) pathway. However, the mismatched base pairs in DNA can occur from a spontaneous conversion of 5-methylcytosine to thymine and DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved special DNA repair systems that target the non-damaged DNA strand in a duplex to remove mismatched regular DNA bases. Mismatch-specific adenine- and thymine-DNA glycosylases (MutY/MUTYH and TDG/MBD4, respectively) initiated BER and mismatch repair (MMR) pathways can recognize and remove normal DNA bases in mismatched DNA duplexes. Importantly, in DNA repair deficient cells bacterial MutY, human TDG and mammalian MMR can act in the aberrant manner: MutY and TDG removes adenine and thymine opposite misincorporated 8-oxoguanine and damaged adenine, respectively, whereas MMR removes thymine opposite to O-methylguanine. These unusual activities lead either to mutations or futile DNA repair, thus indicating that the DNA repair pathways which target non-damaged DNA strand can act in aberrant manner and introduce genome instability in the presence of unrepaired DNA lesions. Evidences accumulated showing that in addition to the accumulation of oxidatively damaged DNA in cells, the aberrant DNA repair can also contribute to cancer, brain disorders and premature senescence. For example, the aberrant BER and MMR pathways for oxidized guanine residues can lead to trinucleotide expansion that underlies Huntington's disease, a severe hereditary neurodegenerative syndrome. This review summarises the present knowledge about the aberrant DNA repair pathways for oxidized base modifications and their possible role in age-related diseases.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.11.040DOI Listing
June 2017

The major Arabidopsis thaliana apurinic/apyrimidinic endonuclease, ARP is involved in the plant nucleotide incision repair pathway.

DNA Repair (Amst) 2016 12 29;48:30-42. Epub 2016 Oct 29.

Groupe «Réparation de l'ADN», Equipe Labellisée par la Ligue Nationale contre le Cancer, CNRS UMR8200, Université Paris-Sud, Gustave Roussy Cancer Campus, F-94805 Villejuif Cedex, France. Electronic address:

Apurinic/apyrimidinic (AP) endonucleases are important DNA repair enzymes involved in two overlapping pathways: DNA glycosylase-initiated base excision (BER) and AP endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, AP endonucleases cleave DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in NIR, the same AP endonucleases incise DNA 5' to a wide variety of oxidized bases. The flowering plant Arabidopsis thaliana contains three genes encoding homologues of major human AP endonuclease 1 (APE1): Arp, Ape1L and Ape2. It has been shown that all three proteins contain AP site cleavage and 3'-repair phosphodiesterase activities; however, it was not known whether the plant AP endonucleases contain the NIR activity. Here, we report that ARP proteins from Arabidopsis and common wheat (Triticum aestivum) contain NIR and 3'→5' exonuclease activities in addition to their AP endonuclease and 3'-repair phosphodiesterase functions. The steady-state kinetic parameters of reactions indicate that Arabidopsis ARP cleaves oligonucleotide duplexes containing α-anomeric 2'-deoxyadenosine (αdA) and 5,6-dihydrouridine (DHU) with efficiencies (k/K=134 and 7.3 μM·min, respectively) comparable to those of the human counterpart. However, the ARP-catalyzed 3'-repair phosphodiesterase and 3'→5' exonuclease activities (k/K=314 and 34 μM·min, respectively) were about 10-fold less efficient as compared to those of APE1. Interestingly, homozygous A. thaliana arp mutant exhibited high sensitivity to methyl methanesulfonate and tert-butyl hydroperoxide, but not to HO, suggesting that ARP is a major plant AP endonuclease that removes abasic sites and specific types of oxidative DNA base damage. Taken together, these data establish the presence of the NIR pathway in plants and suggest its possible role in the repair of DNA damage generated by oxidative stress.
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http://dx.doi.org/10.1016/j.dnarep.2016.10.009DOI Listing
December 2016

8-Oxoguanine Affects DNA Backbone Conformation in the EcoRI Recognition Site and Inhibits Its Cleavage by the Enzyme.

PLoS One 2016 17;11(10):e0164424. Epub 2016 Oct 17.

Chemistry Department, Western Washington University, Bellingham, WA, United States of America.

8-oxoguanine is one of the most abundant and impactful oxidative DNA lesions. However, the reasons underlying its effects, especially those not directly explained by the altered base pairing ability, are poorly understood. We report the effect of the lesion on the action of EcoRI, a widely used restriction endonuclease. Introduction of 8-oxoguanine inside, or adjacent to, the GAATTC recognition site embedded within the Drew-Dickerson dodecamer sequence notably reduced the EcoRI activity. Solution NMR revealed that 8-oxoguanine in the DNA duplex causes substantial alterations in the sugar-phosphate backbone conformation, inducing a BI→BII transition. Moreover, molecular dynamics of the complex suggested that 8-oxoguanine, although does not disrupt the sequence-specific contacts formed by the enzyme with DNA, shifts the distribution of BI/BII backbone conformers. Based on our data, we propose that the disruption of enzymatic cleavage can be linked with the altered backbone conformation and dynamics in the free oxidized DNA substrate and, possibly, at the protein-DNA interface.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0164424PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066940PMC
May 2017

Base excision DNA repair in the embryonic development of the sea urchin, Strongylocentrotus intermedius.

Mol Biosyst 2016 06;12(7):2247-56

Institute of Chemical Biology and Fundamental Medicine SB RAS, 8 Lavrentieva Ave., Novosibirsk 630090, Russia. and Novosibirsk State University, 2 Pirogova St., Novosibirsk 630090, Russia.

In actively proliferating cells, such as the cells of the developing embryo, DNA repair is crucial for preventing the accumulation of mutations and synchronizing cell division. Sea urchin embryo growth was analyzed and extracts were prepared. The relative activity of DNA polymerase, apurinic/apyrimidinic (AP) endonuclease, uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and other glycosylases was analyzed using specific oligonucleotide substrates of these enzymes; the reaction products were resolved by denaturing 20% polyacrylamide gel electrophoresis. We have characterized the profile of several key base excision repair activities in the developing embryos (2 blastomers to mid-pluteus) of the grey sea urchin, Strongylocentrotus intermedius. The uracil-DNA glycosylase specific activity sharply increased after blastula hatching, whereas the specific activity of 8-oxoguanine-DNA glycosylase steadily decreased over the course of the development. The AP-endonuclease activity gradually increased but dropped at the last sampled stage (mid-pluteus 2). The DNA polymerase activity was high at the first cleavage division and then quickly decreased, showing a transient peak at blastula hatching. It seems that the developing sea urchin embryo encounters different DNA-damaging factors early in development within the protective envelope and later as a free-floating larva, with hatching necessitating adaptation to the shift in genotoxic stress conditions. No correlation was observed between the dynamics of the enzyme activities and published gene expression data from developing congeneric species, S. purpuratus. The results suggest that base excision repair enzymes may be regulated in the sea urchin embryos at the level of covalent modification or protein stability.
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http://dx.doi.org/10.1039/c5mb00906eDOI Listing
June 2016

A dynamic checkpoint in oxidative lesion discrimination by formamidopyrimidine-DNA glycosylase.

Nucleic Acids Res 2016 Jan 8;44(2):683-94. Epub 2015 Nov 8.

Department of Chemistry, Stony Brook University, Stony Brook, NY 11794, USA Department of Chemistry, Stony Brook University, Stony Brook, NY 11794, USA

In contrast to proteins recognizing small-molecule ligands, DNA-dependent enzymes cannot rely solely on interactions in the substrate-binding centre to achieve their exquisite specificity. It is widely believed that substrate recognition by such enzymes involves a series of conformational changes in the enzyme-DNA complex with sequential gates favoring cognate DNA and rejecting nonsubstrates. However, direct evidence for such mechanism is limited to a few systems. We report that discrimination between the oxidative DNA lesion, 8-oxoguanine (oxoG) and its normal counterpart, guanine, by the repair enzyme, formamidopyrimidine-DNA glycosylase (Fpg), likely involves multiple gates. Fpg uses an aromatic wedge to open the Watson-Crick base pair and everts the lesion into its active site. We used molecular dynamics simulations to explore the eversion free energy landscapes of oxoG and G by Fpg, focusing on structural and energetic details of oxoG recognition. The resulting energy profiles, supported by biochemical analysis of site-directed mutants disturbing the interactions along the proposed path, show that Fpg selectively facilitates eversion of oxoG by stabilizing several intermediate states, helping the rapidly sliding enzyme avoid full extrusion of every encountered base for interrogation. Lesion recognition through multiple gating intermediates may be a common theme in DNA repair enzymes.
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http://dx.doi.org/10.1093/nar/gkv1092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4737139PMC
January 2016

Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis.

DNA Repair (Amst) 2015 Sep 22;33:1-16. Epub 2015 May 22.

National Center for Biotechnology, Astana 010000, Kazakhstan. Electronic address:

Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Pathogenic bacteria Mycobacterium tuberculosis contains two AP endonucleases: MtbXthA and MtbNfo members of the exonuclease III and endonuclease IV families, which are exemplified by Escherichia coli Xth and Nfo, respectively. It has been shown that both MtbXthA and MtbNfo contain AP endonuclease and 3'→5' exonuclease activities. However, it remains unclear whether these enzymes hold 3'-repair phosphodiesterase and nucleotide incision repair (NIR) activities. Here, we report that both mycobacterial enzymes have 3'-repair phosphodiesterase and 3'-phosphatase, and MtbNfo contains in addition a very weak NIR activity. Interestingly, depending on pH, both enzymes require different concentrations of divalent cations: 0.5mM MnCl2 at pH 7.6 and 10 mM at pH 6.5. MtbXthA requires a low ionic strength and 37 °C, while MtbNfo requires high ionic strength (200 mM KCl) and has a temperature optimum at 60 °C. Point mutation analysis showed that D180 and N182 in MtbXthA and H206 and E129 in MtbNfo are critical for enzymes activities. The steady-state kinetic parameters indicate that MtbXthA removes 3'-blocking sugar-phosphate and 3'-phosphate moieties at DNA strand breaks with an extremely high efficiency (kcat/KM=440 and 1280 μM(-1)∙min(-1), respectively), while MtbNfo exhibits much lower 3'-repair activities (kcat/KM=0.26 and 0.65 μM(-1)∙min(-1), respectively). Surprisingly, both MtbXthA and MtbNfo exhibited very weak AP site cleavage activities, with kinetic parameters 100- and 300-fold lower, respectively, as compared with the results reported previously. Expression of MtbXthA and MtbNfo reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H2O2 to various degrees. Taken together, these data establish the DNA substrate specificity of M. tuberculosis AP endonucleases and suggest their possible role in the repair of oxidative DNA damage generated by endogenous and host- imposed factors.
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http://dx.doi.org/10.1016/j.dnarep.2015.05.007DOI Listing
September 2015

Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III.

J Biol Chem 2015 Jun 13;290(23):14338-49. Epub 2015 Apr 13.

From the Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentyev Ave., Novosibirsk 630090, Russia, the Department of Natural Sciences, Novosibirsk State University, 2 Pirogova St., Novosibirsk 630090, Russia, and

Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3'-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln(41) and Leu(81) as DNA lesion sensors.
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http://dx.doi.org/10.1074/jbc.M114.621128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505503PMC
June 2015

Inhibition of abasic site cleavage in bubble DNA by multifunctional protein YB-1.

J Mol Recognit 2015 Feb 21;28(2):117-23. Epub 2015 Jan 21.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk, 630090, Russia.

Y-box binding protein 1 (YB-1) is widely known to participate in a multiple DNA and RNA processing events in the living cell. YB-1 is also regarded as a putative component of DNA repair. This possibility is supported by relocalization of YB-1 into the nucleus following genotoxic stress. Increased affinity of YB-1 for damaged DNA, especially in its single-stranded form, and its functional interaction with proteins responsible for the initiation of apurinic/apyrimidinic (AP) site repair, namely, AP endonuclease 1 and DNA glycosylase NEIL1, suggest that YB-1 could be involved in the repair of AP sites as a regulatory protein. Here we show that YB-1 has a significant inhibitory effect on the cleavage of AP sites located in single-stranded DNA and in DNA bubble structures. Such interference may be considered as a possible mechanism to prevent single-stranded intermediates of DNA replication, transcription and repair from being converted into highly genotoxic DNA strand breaks, thus allowing the cell to coordinate different DNA processing mechanisms.
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http://dx.doi.org/10.1002/jmr.2435DOI Listing
February 2015

Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition.

Nucleic Acids Res 2015 Jan 17;43(1):272-81. Epub 2014 Dec 17.

Department of Chemistry, Stony Brook University, Stony Brook, NY 11794, USA Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY 11794, USA

Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that Fpg, and possibly other DNA glycosylases, convert part of the binding energy into active destabilization of their substrates, using the energy differences between normal and damaged bases for fast substrate discrimination.
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http://dx.doi.org/10.1093/nar/gku1300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288190PMC
January 2015