Publications by authors named "Inga Grin"

18 Publications

  • Page 1 of 1

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

Mechanisms of Sugar Beet Response to Biotic and Abiotic Stresses.

Adv Exp Med Biol 2020 ;1241:167-194

Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin, China.

Sugar beet is used not only in the sugar production, but also in a wide range of industries including the production of bioethanol as a source of renewable energy, extraction of pectin and production of molasses. The red beetroot has attracted much attention as health-promoting and disease-preventing functional food. The negative effects of environmental stresses, including abiotic and biotic ones, significantly decrease the cash crop sugar beet productivity. In this paper, we outline the mechanisms of sugar beet response to biotic and abiotic stresses at the levels of physiological change, the genes' functions, transcription and translation. Regarding the physiological changes, most research has been carried out on salt and drought stress. The functions of genes from sugar beet in response to salt, cold and heavy metal stresses were mainly investigated by transgenic technologies. At the transcriptional level, the transcriptome analysis of sugar beet in response to salt, cold and biotic stresses were conducted by RNA-Seq or SSH methods. At the translational level, more than 800 differentially expressed proteins in response to salt, K/Na ratio, iron deficiency and resupply and heavy metal (zinc) stress were identified by quantitative proteomics techniques. Understanding how sugar beet respond and tolerate biotic and abiotic stresses is important for boosting sugar beet productivity under these challenging conditions. In order to minimize the negative impact of these stresses, studying how the sugar beet has evolved stress coping mechanisms will provide new insights and lead to novel strategies for improving the breeding of stress-resistant sugar beet and other crops.
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http://dx.doi.org/10.1007/978-3-030-41283-8_10DOI Listing
July 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

Photoinduced inhibition of DNA repair enzymes and the possible mechanism of photochemical transformations of the ruthenium nitrosyl complex [RuNO(β-Pic)(NO)OH].

Metallomics 2019 12;11(12):1999-2009

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

In this work we have demonstrated that the ruthenium nitrosyl complex [RuNO(β-Pic)2(NO2)2OH] is suitable for investigation of the inactivation of DNA repair enzymes in vitro. Photoinduced inhibition of DNA glycosylases such as E. coli Endo III, plant NtROS1, mammalian mNEIL1 and hNEIL2 occurs to an extent of ≥90% after irradiation with the ruthenium complex. The photophysical and photochemical processes of [RuNO(β-Pic)2(NO2)2OH] were investigated using stationary and time-resolved spectroscopy, and mass spectrometry. A possible mechanism of the photo-processes was proposed from the combined spectroscopic study and DTF calculations, which reveal that the photolysis is multistage. The primary and secondary photolysis stages are the photo-induced cleavage of the Ru-NO bond with the formation of a free nitric oxide and RuIII complex followed by ligand exchange with solvent. For E. coli Endo III, covalent interaction with the photolysis product was confirmed by UV-vis and mass spectrometric methods.
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http://dx.doi.org/10.1039/c9mt00153kDOI Listing
December 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

Data set on the synthesis and properties of 2',3'-dideoxyuridine triphosphate conjugated to SiO nanoparticles.

Data Brief 2018 Dec 4;21:540-547. Epub 2018 Oct 4.

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

SiO nanoparticles were used as a transport system for cellular delivery of phosphorylated 2',3'-dideoxyuridine to increase its anticancer potency. This data set is related to the research article entitled "2',3'-Dideoxyuridine triphosphate conjugated to SiO nanoparticles: synthesis and evaluation of antiproliferative activity" (Vasilyeva et al., 2018) [1]. It includes a protocol for the synthesis of 2',3'-dideoxyuridine-5'-{N-[4-(prop-2-yn-1-yloxy)butyl]-γ-amino}-triphosphate, its identification by NMR, IR and ESI-MS, experimental procedure of covalent attachment to SiO nanoparticles with Cu-catalyzed click-chemistry, experimental data on chemical stability of the conjugate at different pH values and cytotoxicity assessment of antiproliferative effect of the conjugate.
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http://dx.doi.org/10.1016/j.dib.2018.09.127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199781PMC
December 2018

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

2',3'-Dideoxyuridine triphosphate conjugated to SiO nanoparticles: Synthesis and evaluation of antiproliferative activity.

Bioorg Med Chem Lett 2018 04 7;28(7):1248-1251. Epub 2018 Feb 7.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 8 Lavrentiev Avenue, Novosibirsk 630090, Russia.

A conjugate of triphosphorylated 2',3'-dideoxyuridine (ddU) with SiO nanoparticles was obtained via the CuAAC click chemistry between a γ-alkynyl ddU triphosphate and azido-modified SiO nanoparticles. Assessment of cytotoxicity in human breast adenocarcinoma MCF7 cells demonstrated that ddU triphosphate conjugated to SiO nanoparticles exhibited a 50% decrease in cancer cell growth at a concentration of 183 ± 57 µg/mL, which corresponds to 22 ± 7 µM of the parent nucleotide, whereas the parent nucleoside, nucleotide and alkynyl triphosphate precursor do not show any cytotoxicity. The data provide an example of remarkable potential of novel conjugates of SiO nanoparticles with phosphorylated nucleoside analogues, even those, which have not been used previously as therapeutics, for application as new anticancer agents.
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http://dx.doi.org/10.1016/j.bmcl.2018.02.007DOI Listing
April 2018

Structural comparison of AP endonucleases from the exonuclease III family reveals new amino acid residues in human AP endonuclease 1 that are involved in incision of damaged DNA.

Biochimie 2016 Sep-Oct;128-129:20-33. Epub 2016 Jun 22.

Institute for Integrative Biology of the Cell (I2BC), CNRS CEA Univ Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette 91198, France. Electronic address:

Oxidatively damaged DNA bases are substrates for two overlapping repair pathways: DNA glycosylase-initiated base excision repair (BER) and apurinic/apyrimidinic (AP) endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in the NIR pathway, the same AP endonuclease incises DNA 5' to an oxidized base. The majority of characterized AP endonucleases possess classic BER activities, and approximately a half of them can also have a NIR activity. At present, the molecular mechanism underlying DNA substrate specificity of AP endonucleases remains unclear mainly due to the absence of a published structure of the enzyme in complex with a damaged base. To identify critical residues involved in the NIR function, we performed biochemical and structural characterization of Bacillus subtilis AP endonuclease ExoA and compared its crystal structure with the structures of other AP endonucleases: Escherichia coli exonuclease III (Xth), human APE1, and archaeal Mth212. We found conserved amino acid residues in the NIR-specific enzymes APE1, Mth212, and ExoA. Four of these positions were studied by means of point mutations in APE1: we applied substitution with the corresponding residue found in NIR-deficient E. coli Xth (Y128H, N174Q, G231S, and T268D). The APE1-T268D mutant showed a drastically decreased NIR activity and an inverted Mg(2+) dependence of the AP site cleavage activity, which is in line with the presence of an aspartic residue at the equivalent position among other known NIR-deficient AP endonucleases. Taken together, these data show that NIR is an evolutionarily conserved function in the Xth family of AP endonucleases.
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http://dx.doi.org/10.1016/j.biochi.2016.06.011DOI Listing
January 2017

An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation.

Nucleic Acids Res 2016 05 3;44(8):3713-27. Epub 2016 Feb 3.

Laboratoire «Stabilité Génétique et Oncogenèse» CNRS, UMR 8200, Univ. Paris-Sud, Université Paris-Saclay, Equipe Labellisée Ligue Contre le Cancer, F-94805 Villejuif, France Gustave Roussy Cancer Campus, F-94805 Villejuif, France

Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM.
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http://dx.doi.org/10.1093/nar/gkw059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856981PMC
May 2016

Functional variants of human APE1 rescue the DNA repair defects of the yeast AP endonuclease/3'-diesterase-deficient strain.

DNA Repair (Amst) 2014 Oct 9;22:53-66. Epub 2014 Aug 9.

Maisonneuve-Rosemont Hospital, Research Center, Université de Montréal 5415 Boul. de l' Assomption, Montréal, Québec, Canada H1T 2M4. Electronic address:

Human APE1 is an essential enzyme performing functions in DNA repair and transcription. It possesses four distinct repair activities acting on a variety of base and sugar derived DNA lesions. APE1 has seven cysteine residues and Cys65, and to a lesser extent Cys93 and Cys99, is uniquely involved in maintaining a subset of transcription factors in the reduced and active state. Four of the cysteines Cys93, 99, 208 and 310 of APE1 are located proximal to its active site residues Glu96, Asp210 and His309 involved in processing damaged DNA, raising the possibility that missense mutation of these cysteines could alter the enzyme DNA repair functions. An earlier report documented that serine substitution of the individual cysteine residues did not affect APE1 ability to cleave an abasic site oligonucleotide substrate in vitro, except for Cys99Ser, although any consequences of these variants in the repair of in vivo DNA lesions were not tested. Herein, we mutated all seven cysteines of APE1, either singly or in combination, to alanine and show that none of the resulting variants interfered with the enzyme DNA repair functions. Cross-specie complementation analysis reveals that these APE1 cysteine variants fully rescued the yeast DNA repair deficient strain YW778, lacking AP endonucleases and 3'-diesterases, from toxicities caused by DNA damaging agents. Moreover, the elevated spontaneous mutations arising in strain YW778 from the lack of the DNA repair activities were completely suppressed by the APE1 cysteine variants. These findings suggest that the cysteine residues of APE1 are unlikely to play a role in the DNA repair functions of the enzyme in vivo. We also examine other APE1 missense mutations and provide the first evidence that the variant Asp308Ala with normal AP endonuclease, but devoid of 3'→5' exonuclease, displays hypersensitivity to the anticancer drug bleomycin, and not to other agents, suggesting that it has a defect in processing unique DNA lesions. Molecular modeling reveals that Asp308Ala cannot make proper contact with Mg(2+) and may alter the enzyme ability to cleave or disassociate from specific DNA lesions.
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http://dx.doi.org/10.1016/j.dnarep.2014.07.010DOI Listing
October 2014

Cloning and characterization of a wheat homologue of apurinic/apyrimidinic endonuclease Ape1L.

PLoS One 2014 25;9(3):e92963. Epub 2014 Mar 25.

Groupe «Réparation de l'ADN», CNRS UMR8200, Université Paris-Sud, Institut Gustave Roussy, Villejuif, France.

Background: Apurinic/apyrimidinic (AP) endonucleases are key DNA repair enzymes involved in the base excision repair (BER) pathway. In BER, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases and/or oxidative damage. A Triticum aestivum cDNA encoding for a putative homologue of ExoIII family AP endonucleases which includes E. coli Xth, human APE1 and Arabidopsis thaliana AtApe1L has been isolated and its protein product purified and characterized.

Methodology/principal Findings: We report that the putative wheat AP endonuclease, referred here as TaApe1L, contains AP endonuclease, 3'-repair phosphodiesterase, 3'-phosphatase and 3' → 5' exonuclease activities. Surprisingly, in contrast to bacterial and human AP endonucleases, addition of Mg(2+) and Ca(2+) (5-10 mM) to the reaction mixture inhibited TaApe1L whereas the presence of Mn(2+), Co(2+) and Fe(2+) cations (0.1-1.0 mM) strongly stimulated all its DNA repair activities. Optimization of the reaction conditions revealed that the wheat enzyme requires low divalent cation concentration (0.1 mM), mildly acidic pH (6-7), low ionic strength (20 mM KCl) and has a temperature optimum at around 20 °C. The steady-state kinetic parameters of enzymatic reactions indicate that TaApe1L removes 3'-blocking sugar-phosphate and 3'-phosphate groups with good efficiency (kcat/KM = 630 and 485 μM(-1) · min(-1), respectively) but possesses a very weak AP endonuclease activity as compared to the human homologue, APE1.

Conclusions/significance: Taken together, these data establish the DNA substrate specificity of the wheat AP endonuclease and suggest its possible role in the repair of DNA damage generated by endogenous and environmental factors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092963PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965494PMC
January 2015

Excision of 8-oxoguanine from methylated CpG dinucleotides by human 8-oxoguanine DNA glycosylase.

FEBS Lett 2013 Sep 13;587(18):3129-34. Epub 2013 Aug 13.

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

CpG dinucleotides are targets for epigenetic methylation, many of them bearing 5-methylcytosine (mCyt) in the human genome. Guanine in this context can be easily oxidized to 8-oxoguanine (oxoGua), which is repaired by 8-oxoguanine-DNA glycosylase (OGG1). We have studied how methylation affects the efficiency of oxoGua excision from damaged CpG dinucleotides. Methylation of the adjacent cytosine moderately decreased the oxoGua excision rate while methylation opposite oxoGua lowered the rate of product release. Cytosine methylation abolished stimulation of OGG1 by repair endonuclease APEX1. The OGG1 S326C polymorphic variant associated with lung cancer showed poorer base excision and lost sensitivity to the opposite-base methylation. The overall repair in the system reconstituted from purified proteins decreased for CpG with mCyt in the damaged strand.
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http://dx.doi.org/10.1016/j.febslet.2013.08.008DOI Listing
September 2013

Biochemical and structural characterization of the glycosylase domain of MBD4 bound to thymine and 5-hydroxymethyuracil-containing DNA.

Nucleic Acids Res 2012 Oct 30;40(19):9917-26. Epub 2012 Jul 30.

Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette Cedex, F-91198, France.

Active DNA demethylation in mammals occurs via hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) by the ten-eleven translocation family of proteins (TETs). 5hmC residues in DNA can be further oxidized by TETs to 5-carboxylcytosines and/or deaminated by the Activation Induced Deaminase/Apolipoprotein B mRNA-editing enzyme complex family proteins to 5-hydromethyluracil (5hmU). Excision and replacement of these intermediates is initiated by DNA glycosylases such as thymine-DNA glycosylase (TDG), methyl-binding domain protein 4 (MBD4) and single-strand specific monofunctional uracil-DNA glycosylase 1 in the base excision repair pathway. Here, we report detailed biochemical and structural characterization of human MBD4 which contains mismatch-specific TDG activity. Full-length as well as catalytic domain (residues 426-580) of human MBD4 (MBD4(cat)) can remove 5hmU when opposite to G with good efficiency. Here, we also report six crystal structures of human MBD4(cat): an unliganded form and five binary complexes with duplex DNA containing a T•G, 5hmU•G or AP•G (apurinic/apyrimidinic) mismatch at the target base pair. These structures reveal that MBD4(cat) uses a base flipping mechanism to specifically recognize thymine and 5hmU. The recognition mechanism of flipped-out 5hmU bases in MBD4(cat) active site supports the potential role of MBD4, together with TDG, in maintenance of genome stability and active DNA demethylation in mammals.
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http://dx.doi.org/10.1093/nar/gks714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3479182PMC
October 2012

Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1.

J Mol Recognit 2012 Apr;25(4):224-33

Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Prospect Lavrentieva 8, Novosibirsk, 630090, Russia.

DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP site was located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1.
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http://dx.doi.org/10.1002/jmr.2182DOI Listing
April 2012

The role of mammalian NEIL1 protein in the repair of 8-oxo-7,8-dihydroadenine in DNA.

FEBS Lett 2010 Apr 7;584(8):1553-7. Epub 2010 Mar 7.

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

8-oxo-7,8-dihydroadenine (8-oxoAde) is a major product of adenine modification by reactive oxygen species. So far, only one mammalian DNA glycosylase, 8-oxoguanine-DNA-glycosylase 1 (OGG1), has been shown to excise 8-oxoAde, exclusively from pairs with Cyt. We have found that endonuclease VIII-like protein 1 (NEIL1), a mammalian homolog of bacterial endonuclease VIII, can efficiently remove 8-oxoAde from 8-oxoAde:Cyt pairs but not from other contexts. In an in vitro reconstituted system, reactions containing OGG1 produced a fully repaired product, whereas NEIL1 caused an abortive initiation of repair, stopping after 8-oxoAde removal and DNA strand cleavage. This block was partially relieved by polynucleotide kinase/3'-phosphatase. Thus, two alternative routes of 8-oxoAde repair may exist in mammals.
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http://dx.doi.org/10.1016/j.febslet.2010.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3004018PMC
April 2010

Inactivation of NEIL2 DNA glycosylase by pyridoxal phosphate reveals a loop important for substrate binding.

Biochem Biophys Res Commun 2010 Mar 20;394(1):100-5. Epub 2010 Feb 20.

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

Pyridoxal-5'-phosphate (PLP), in addition to its known metabolic functions, inactivates many DNA-dependent enzymes through conjugation to their critical amino groups. We have investigated the ability of PLP to inhibit bifunctional DNA repair glycosylases, which possess a catalytic amine. Of six enzymes tested, only endonuclease VIII-like protein 2 (NEIL2) was significantly inhibited by PLP. The inhibition was due to Schiff base formation between PLP and the enzyme. PLP-conjugated NEIL2 completely lost its ability to bind damaged DNA. Liquid chromatography/nanoelectrospray ionization tandem mass spectrometry of the products of proteolysis of pyridoxylated NEIL2 identified Lys50 as the site of modification. Thus, the beta2/beta3 loop where Lys50 is located in NEIL2 is important for DNA binding, presumably lies next to a phosphate-binding site, and may represent a target for regulation of the enzyme activity.
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http://dx.doi.org/10.1016/j.bbrc.2010.02.121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857644PMC
March 2010

Deoxyribophosphate lyase activity of mammalian endonuclease VIII-like proteins.

FEBS Lett 2006 Sep 15;580(20):4916-22. Epub 2006 Aug 15.

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

Base excision repair (BER) protects cells from nucleobase DNA damage. In eukaryotic BER, DNA glycosylases generate abasic sites, which are then converted to deoxyribo-5'-phosphate (dRP) and excised by a dRP lyase (dRPase) activity of DNA polymerase beta (Polbeta). Here, we demonstrate that NEIL1 and NEIL2, mammalian homologs of bacterial endonuclease VIII, excise dRP by beta-elimination with the efficiency similar to Polbeta. DNA duplexes imitating BER intermediates after insertion of a single nucleotide were better substrates. NEIL1 and NEIL2 supplied dRPase activity in BER reconstituted with dRPase-null Polbeta. Our results suggest a role for NEILs as backup dRPases in mammalian cells.
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http://dx.doi.org/10.1016/j.febslet.2006.08.011DOI Listing
September 2006