Publications by authors named "Olga I Lavrik"

96 Publications

Discovery of Novel Sultone Fused Berberine Derivatives as Promising Tdp1 Inhibitors.

Molecules 2021 Mar 30;26(7). Epub 2021 Mar 30.

N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, 9, Akademika Lavrentieva Ave., 630090 Novosibirsk, Russia.

A new type of berberine derivatives was obtained by the reaction of berberrubine with aliphatic sulfonyl chlorides. The new polycyclic compounds have a sultone ring condensed to and rings of a protoberberine core. The reaction conditions were developed to facilitate the formation of sultones with high yields without by-product formation. Thus, it was shown that the order of addition of reagents affects the composition of the reaction products: when sulfochlorides are added to berberrubine, their corresponding 9--sulfonates are predominantly formed; when berberrubine is added to pre-generated sulfenes, sultones are the only products. The reaction was shown to proceed stereo-selectively and the cycle configuration was confirmed by 2D NMR spectroscopy. The inhibitory activity of the synthesized sultones and their 12-brominated analogs against the DNA-repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1), an important target for a potential antitumor therapy, was studied. All derivatives were active in the micromolar and submicromolar range, in contrast to the acyclic analogs and 9--sulfonates, which were inactive. The significance of the sultone cycle and bromine substituent in binding with the enzyme was confirmed using molecular modeling. The active inhibitors are mostly non-toxic to the HeLa cancer cell line, and several ligands show synergy with topotecan, a topoisomerase 1 poison in clinical use. Thus, novel berberine derivatives can be considered as candidates for adjuvant therapy against cancer.
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http://dx.doi.org/10.3390/molecules26071945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037669PMC
March 2021

Discovery of Novel Sultone Fused Berberine Derivatives as Promising Tdp1 Inhibitors.

Molecules 2021 Mar 30;26(7). Epub 2021 Mar 30.

N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, 9, Akademika Lavrentieva Ave., 630090 Novosibirsk, Russia.

A new type of berberine derivatives was obtained by the reaction of berberrubine with aliphatic sulfonyl chlorides. The new polycyclic compounds have a sultone ring condensed to and rings of a protoberberine core. The reaction conditions were developed to facilitate the formation of sultones with high yields without by-product formation. Thus, it was shown that the order of addition of reagents affects the composition of the reaction products: when sulfochlorides are added to berberrubine, their corresponding 9--sulfonates are predominantly formed; when berberrubine is added to pre-generated sulfenes, sultones are the only products. The reaction was shown to proceed stereo-selectively and the cycle configuration was confirmed by 2D NMR spectroscopy. The inhibitory activity of the synthesized sultones and their 12-brominated analogs against the DNA-repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1), an important target for a potential antitumor therapy, was studied. All derivatives were active in the micromolar and submicromolar range, in contrast to the acyclic analogs and 9--sulfonates, which were inactive. The significance of the sultone cycle and bromine substituent in binding with the enzyme was confirmed using molecular modeling. The active inhibitors are mostly non-toxic to the HeLa cancer cell line, and several ligands show synergy with topotecan, a topoisomerase 1 poison in clinical use. Thus, novel berberine derivatives can be considered as candidates for adjuvant therapy against cancer.
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http://dx.doi.org/10.3390/molecules26071945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037669PMC
March 2021

Strand Displacement Activity of PrimPol.

Int J Mol Sci 2020 Nov 27;21(23). Epub 2020 Nov 27.

Institute of Molecular Genetics, National Research Center "Kurchatov Institute", Kurchatov sq. 2, 123182 Moscow, Russia.

Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and possesses the conditional strand displacement activity stimulated by Mn ions and accessory replicative proteins RPA and PolDIP2. The DNA displacement activity of PrimPol was found to be more efficient than the RNA displacement activity and FEN1 processed the 5'-DNA flaps generated by PrimPol in vitro.
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http://dx.doi.org/10.3390/ijms21239027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729601PMC
November 2020

Deoxycholic acid as a molecular scaffold for tyrosyl-DNA phosphodiesterase 1 inhibition: A synthesis, structure-activity relationship and molecular modeling study.

Steroids 2021 Jan 19;165:108771. Epub 2020 Nov 19.

N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, SB RAS, 9, Lavrent'ev Ave., Novosibirsk 630090, Russian Federation.

Para-Bromoanilides of deoxycholic acid with various functional groups on the steroid scaffold were designed as promising tyrosyl-DNA phosphodiesterase 1 (Tdp1) inhibitors. Tdp1 is a DNA repair enzyme, involved in removing DNA damage caused by topoisomerase I poisons; an important class of anticancer drugs. Thus, reducing the activity of Tdp1 can increase the efficacy of anticancer drugs in current use. Inhibitory activity in the low micromolar and submicromolar concentrations was observed with 3,12-dimethoxy para-bromoanilide 17 being the most active with an IC value of 0.27 μM. The activity of N-methyl para-bromoanilides was 3-4.8 times lower than of the corresponding para-bromoanilides. Increased potency of the ligands was seen with higher molecular weight and log P values. The ligands were evaluated for their cytotoxic potential in a panel of tumor cell lines; all were nontoxic to the A549 pulmonary adenocarcinoma cell line. However, derivatives containing a hydroxyl group at the 12th position were more toxic than their 12-hydroxyl group counterparts (acetoxy-, oxo- and methoxy- group) against HCT-116 human colon and HepG2 hepatocellular carcinomas. In addition, an N-methyl substitution led to an increase in toxicity for the HCT-116 and HepG2 cell lines. The excellent activity as well as low cytotoxicity, derivative 17 can be considered as a lead compound for further development.
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http://dx.doi.org/10.1016/j.steroids.2020.108771DOI Listing
January 2021

PARPs' impact on base excision DNA repair.

Authors:
Olga I Lavrik

DNA Repair (Amst) 2020 09;93:102911

Institute of Chemical Biology and Fundamental Medicine (ICBFM), Lavrentiev Ave. 8, Novosibirsk 630090, Russia; Novosibirsk State University, Novosibirsk, 630090, Russia. Electronic address:

Poly(ADP-ribosyl)ation is one of immediate cellular responses to DNA damage and is catalyzed by poly(ADP-ribose) polymerases (PARPs). PARP1 is a well-known regulator of DNA repair. Another member of this family, PARP2, was discovered later. The study of PARP1 and PARP2 functions started a long time ago, and special attention has been given to the role of these enzymes in base excision repair. This review summarizes my lab's data on the functions of PARP1 and PARP2 in base excision repair as well as the results obtained in the course of our collaboration with Dr. Samuel H. Wilson.
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http://dx.doi.org/10.1016/j.dnarep.2020.102911DOI Listing
September 2020

The First Berberine-Based Inhibitors of Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), an Important DNA Repair Enzyme.

Int J Mol Sci 2020 Sep 28;21(19). Epub 2020 Sep 28.

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

A series of berberine and tetrahydroberberine sulfonate derivatives were prepared and tested against the tyrosyl-DNA phosphodiesterase 1 (Tdp1) DNA-repair enzyme. The berberine derivatives inhibit the Tdp1 enzyme in the low micromolar range; this is the first reported berberine based Tdp1 inhibitor. A structure-activity relationship analysis revealed the importance of bromine substitution in the 12-position on the tetrahydroberberine scaffold. Furthermore, it was shown that the addition of a sulfonate group containing a polyfluoroaromatic moiety at position 9 leads to increased potency, while most of the derivatives containing an alkyl fragment at the same position were not active. According to the molecular modeling, the bromine atom in position 12 forms a hydrogen bond to histidine 493, a key catalytic residue. The cytotoxic effect of topotecan, a clinically important topoisomerase 1 inhibitor, was doubled in the cervical cancer HeLa cell line by derivatives 11g and 12g; both displayed low toxicity without topotecan. Derivatives 11g and 12g can therefore be used for further development to sensitize the action of clinically relevant Topo1 inhibitors.
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http://dx.doi.org/10.3390/ijms21197162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582571PMC
September 2020

Fused in Sarcoma (FUS) in DNA Repair: Tango with Poly(ADP-ribose) Polymerase 1 and Compartmentalisation of Damaged DNA.

Int J Mol Sci 2020 Sep 24;21(19). Epub 2020 Sep 24.

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

The fused in sarcoma (FUS) protein combines prion-like properties with a multifunctional DNA/RNA-binding domain and has functions spanning the regulation of RNA metabolism, including transcription, pre-mRNA splicing, mRNA transport and translation. In addition to its roles in RNA metabolism, FUS is implicated in the maintenance of DNA integrity. In this review, we examine the participation of FUS in major DNA repair pathways, focusing on DNA repair associated with poly(ADP-ribosyl)ation events and on how the interaction of FUS with poly(ADP-ribose) may orchestrate transient compartmentalisation of DNA strand breaks. Unravelling how prion-like RNA-binding proteins control DNA repair pathways will deepen our understanding of the pathogenesis of some neurological diseases and cancer as well as provide the basis for the development of relevant innovative therapeutic technologies. This knowledge may also extend the range of applications of poly(ADP-ribose) polymerase inhibitors to the treatment of neurodegenerative diseases related to RNA-binding proteins in the cell, e.g., amyotrophic lateral sclerosis and frontotemporal lobar degeneration.
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http://dx.doi.org/10.3390/ijms21197020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582374PMC
September 2020

Regulation of Poly(ADP-Ribose) Polymerase 1 Activity by Y-Box-Binding Protein 1.

Biomolecules 2020 09 16;10(9). Epub 2020 Sep 16.

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

Y-box-binding protein 1 (YB-1) is a multifunctional positively charged protein that interacts with DNA or RNA and poly(ADP-ribose) (PAR). YB-1 is poly(ADP-ribosyl)ated and stimulates poly(ADP-ribose) polymerase 1 (PARP1) activity. Here, we studied the mechanism of YB-1-dependent PAR synthesis by PARP1 in vitro using biochemical and atomic force microscopy assays. PAR synthesis activity of PARP1 is known to be facilitated by co-factors such as Mg. However, in contrast to an Mg-dependent reaction, the activation of PARP1 by YB-1 is accompanied by overall up-regulation of protein PARylation and shortening of the PAR polymer. Therefore, YB-1 and cation co-factors stimulated PAR synthesis in divergent ways. PARP1 autoPARylation in the presence of YB-1 as well as trans-PARylation of YB-1 are greatly affected by the type of damaged DNA, suggesting that PARP1 activation depends on the formation of a PARP1-YB-1-DNA ternary complex. An unstructured C-terminal part of YB-1 involved in an interaction with PAR behaves similarly to full-length YB-1, indicating that both DNA and PAR binding are involved in the stimulation of PARP1 activity by YB-1. Thus, YB-1 is likely linked to the regulation of PARylation events in cells via an interaction with PAR and damaged DNA.
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http://dx.doi.org/10.3390/biom10091325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565162PMC
September 2020

Inhibition of Tyrosyl-DNA Phosphodiesterase 1 by Lipophilic Pyrimidine Nucleosides.

Molecules 2020 Aug 13;25(16). Epub 2020 Aug 13.

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova Str., 119991 Moscow, Russia.

Inhibition of DNA repair enzymes tyrosyl-DNA phosphodiesterase 1 and poly(ADP-ribose)polymerases 1 and 2 in the presence of pyrimidine nucleoside derivatives was studied here. New effective Tdp1 inhibitors were found in a series of nucleoside derivatives possessing 2',3',5'-tri--benzoyl-d-ribofuranose and 5-substituted uracil moieties and have half-maximal inhibitory concentrations (IC) in the lower micromolar and submicromolar range. 2',3',5'-Tri--benzoyl-5-iodouridine manifested the strongest inhibitory effect on Tdp1 (IC = 0.6 μM). A decrease in the number of benzoic acid residues led to a marked decline in the inhibitory activity, and pyrimidine nucleosides lacking lipophilic groups (uridine, 5-fluorouridine, 5-chlorouridine, 5-bromouridine, 5-iodouridine, and ribothymidine) did not cause noticeable inhibition of Tdp1 (IC > 50 μM). No PARP1/2 inhibitors were found among the studied compounds (residual activity in the presence of 1 mM substances was 50-100%). Several -benzoylated uridine and cytidine derivatives strengthened the action of topotecan on HeLa cervical cancer cells.
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http://dx.doi.org/10.3390/molecules25163694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465190PMC
August 2020

Design, Synthesis, and Biological Investigation of Novel Classes of 3-Carene-Derived Potent Inhibitors of TDP1.

Molecules 2020 Jul 31;25(15). Epub 2020 Jul 31.

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

Two novel structural types of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors with hexahydroisobenzofuran and 3-oxabicyclo [3.3.1]nonane scaffolds were discovered. These monoterpene-derived compounds were synthesized through preliminary isomerization of (+)-3-carene to (+)-2-carene followed by reaction with heteroaromatic aldehydes. All the compounds inhibit the TDP1 enzyme at micro- and submicromolar levels, with the most potent compound having an IC value of 0.65 μM. TDP1 is an important DNA repair enzyme and a promising target for the development of new chemosensitizing agents. A panel of isogenic clones of the HEK293FT cell line knockout for the gene was created using the CRISPR-Cas9 system. Cytotoxic effects of topotecan (Tpc) and non-cytotoxic compounds of the new structures were investigated separately and jointly in the gene knockout cells. For two TDP1 inhibitors, and , a synergistic effect was observed with Tpc in the HEK293FT cells but was not found in TDP1 -/- cells. Thus, it is likely that the synergistic effect is caused by inhibition of TDP1. Synergy was also found for in other cancer cell lines. Thus, sensitizing cancer cells using a non-cytotoxic drug can enhance the efficacy of currently used pharmaceuticals and, concomitantly, reduce toxic side effects.
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http://dx.doi.org/10.3390/molecules25153496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7436013PMC
July 2020

Photoreactive DNA as a Tool to Study Replication Protein A Functioning in DNA Replication and Repair.

Photochem Photobiol 2020 03 5;96(2):440-449. Epub 2020 Mar 5.

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

Replication protein A (RPA), eukaryotic single-stranded DNA-binding protein, is a key player in multiple processes of DNA metabolism including DNA replication, recombination and DNA repair. Human RPA composed of subunits of 70-, 32- and 14-kDa binds ssDNA with high affinity and interacts specifically with multiple proteins. The RPA heterotrimer binds ssDNA in several modes, with occlusion lengths of 8-10, 13-22 and 30 nucleotides corresponding to global, transitional and elongated conformations of protein. Varying the structure of photoreactive DNA, the intermediates of different stages of DNA replication or DNA repair were designed and applied to identify positioning of the RPA subunits on the specific DNA structures. Using this approach, RPA interactions with various types of DNA structures attributed to replication and DNA repair intermediates were examined. This review is dedicated to blessed memory of Prof. Alain Favre who contributed to the development of photoreactive nucleotide derivatives and their application for the study of protein-nucleic acids interactions.
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http://dx.doi.org/10.1111/php.13222DOI Listing
March 2020

Inhibition of Transcription Induces Phosphorylation of YB-1 at Ser102 and Its Accumulation in the Nucleus.

Cells 2019 Dec 31;9(1). Epub 2019 Dec 31.

Institute of Protein Research, Russian Academy of Sciences, Pushchino 142290, Russia.

The Y-box binding protein 1 (YB-1) is an RNA/DNA-binding protein regulating gene expression in the cytoplasm and the nucleus. Although mostly cytoplasmic, YB-1 accumulates in the nucleus under stress conditions. Its nuclear localization is associated with aggressiveness and multidrug resistance of cancer cells, which makes the understanding of the regulatory mechanisms of YB-1 subcellular distribution essential. Here, we report that inhibition of RNA polymerase II (RNAPII) activity results in the nuclear accumulation of YB-1 accompanied by its phosphorylation at Ser102. The inhibition of kinase activity reduces YB-1 phosphorylation and its accumulation in the nucleus. The presence of RNA in the nucleus is shown to be required for the nuclear retention of YB-1. Thus, the subcellular localization of YB-1 depends on its post-translational modifications (PTMs) and intracellular RNA distribution.
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http://dx.doi.org/10.3390/cells9010104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016903PMC
December 2019

Design, Synthesis and Molecular Modeling Study of Conjugates of ADP and Morpholino Nucleosides as A Novel Class of Inhibitors of PARP-1, PARP-2 and PARP-3.

Int J Mol Sci 2019 Dec 27;21(1). Epub 2019 Dec 27.

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

We report on the design, synthesis and molecular modeling study of conjugates of adenosine diphosphate (ADP) and morpholino nucleosides as potential selective inhibitors of poly(ADP-ribose)polymerases-1, 2 and 3. Sixteen dinucleoside pyrophosphates containing natural heterocyclic bases as well as 5-haloganeted pyrimidines, and mimicking a main substrate of these enzymes, nicotinamide adenine dinucleotide (NAD+)-molecule, have been synthesized in a high yield. Morpholino nucleosides have been tethered to the β-phosphate of ADP via a phosphoester or phosphoramide bond. Screening of the inhibiting properties of these derivatives on the autopoly(ADP-ribosyl)ation of PARP-1 and PARP-2 has shown that the effect depends upon the type of nucleobase as well as on the linkage between ADP and morpholino nucleoside. The 5-iodination of uracil and the introduction of the P-N bond in NAD+-mimetics have shown to increase inhibition properties. Structural modeling suggested that the P-N bond can stabilize the pyrophosphate group in active conformation due to the formation of an intramolecular hydrogen bond. The most active NAD+ analog against PARP-1 contained 5-iodouracil 2'-aminomethylmorpholino nucleoside with IC50 126 ± 6 μM, while in the case of PARP-2 it was adenine 2'-aminomethylmorpholino nucleoside (IC50 63 ± 10 μM). In silico analysis revealed that thymine and uracil-based NAD+ analogs were recognized as the NAD+-analog that targets the nicotinamide binding site. On the contrary, the adenine 2'-aminomethylmorpholino nucleoside-based NAD+ analogs were predicted to identify as PAR-analogs that target the acceptor binding site of PARP-2, representing a novel molecular mechanism for selective PARP inhibition. This discovery opens a new avenue for the rational design of PARP-1/2 specific inhibitors.
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http://dx.doi.org/10.3390/ijms21010214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982223PMC
December 2019

Promising New Inhibitors of Tyrosyl-DNA Phosphodiesterase I (Tdp 1) Combining 4-Arylcoumarin and Monoterpenoid Moieties as Components of Complex Antitumor Therapy.

Int J Mol Sci 2019 Dec 23;21(1). Epub 2019 Dec 23.

Novosibirsk Institute of Chemical Biology and Fundamental Medicine, 8, acad. Lavrentjev ave., 630090 Novosibirsk, Russia.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme in humans, and a current and promising inhibition target for the development of new chemosensitizing agents due to its ability to remove DNA damage caused by topoisomerase 1 (Top1) poisons such as topotecan and irinotecan. Herein, we report our work on the synthesis and characterization of new Tdp1 inhibitors that combine the arylcoumarin (neoflavonoid) and monoterpenoid moieties. Our results showed that they are potent Tdp1 inhibitors with IC values in the submicromolar range. In vivo experiments with mice revealed that compound (IC 0.62 µM) induced a significant increase in the antitumor effect of topotecan on the Krebs-2 ascites tumor model. Our results further strengthen the argument that Tdp1 is a druggable target with the potential to be developed into a clinically-potent adjunct therapy in conjunction with Top1 poisons.
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http://dx.doi.org/10.3390/ijms21010126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982354PMC
December 2019

Human apurinic/apyrimidinic endonuclease 1 is modified in vitro by poly(ADP-ribose) polymerase 1 under control of the structure of damaged DNA.

Biochimie 2020 Jan 24;168:144-155. Epub 2019 Oct 24.

Institute of Chemical Biology and Fundamental Medicine (ICBFM), Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia. Electronic address:

Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential multifunctional protein in mammals involved in base excision DNA repair (BER), regulation of gene expression and RNA metabolism. Its major enzymatic function is incision of AP sites. Poly(ADP-ribose) polymerase 1 (PARP1) modifies itself and target proteins with poly(ADP-ribose) (PAR), contributing to regulation of many processes. To understand molecular basis of functional cooperation between APE1 and PARP1 in BER, we examined PAR-binding activity and ADP-ribosylation of human APE1 in comparison with known targets of PARP1, using the full-length, N-terminally truncated and catalytically inactive forms of APE1. The protein binds preferentially large ADP-ribose polymers, being very similar to DNA polymerase β (Polβ) but contrasting with the scaffold XRCC1 protein. The interaction with PAR involves the universally conserved catalytic portion and the eukaryote-specific extension of APE1. The ADP-ribosylation of APE1 depends on the structure of PARP1-activating DNA, contrasting APE1 with Polβ and XRCC1. Relative levels of APE1 modification in the presence of different DNA substrates were found to correlate with affinities of the DNAs for APE1 and substrate activities in the enzymatic incision, suggesting the ADP-ribosylation to occur within the DNA-mediated ternary complex. This conclusion was confirmed by importance of the length of DNA region 3' to the AP site for the modification. Deletion of the N-terminal extension of APE1 produced no significant influence on both the ADP-ribosylation efficiency and hydrolytic stability of the modified protein, suggesting localization of target amino acids in the conserved catalytic portion. The most efficient ADP-ribosylation of the catalytically inactive APE1 mutant was shown to reduce the level of PARP1 automodification, suggesting possible role of APE1 in modulating PARP1 activity. Our data on primary role of DNA in controlling the PARP-catalysed modification provide new insights into mechanisms of protein targeting for ADP-ribosylation.
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http://dx.doi.org/10.1016/j.biochi.2019.10.011DOI Listing
January 2020

New Hydrazinothiazole Derivatives of Usnic Acid as Potent Tdp1 Inhibitors.

Molecules 2019 Oct 15;24(20). Epub 2019 Oct 15.

N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, 9, Akademika Lavrentieva Ave., Novosibirsk 630090, Russia.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a promising therapeutic target in cancer therapy. Combination chemotherapy using Tdp1 inhibitors as a component can potentially improve therapeutic response to many chemotherapeutic regimes. A new set of usnic acid derivatives with hydrazonothiazole pharmacophore moieties were synthesized and evaluated as Tdp1 inhibitors. Most of these compounds were found to be potent inhibitors with IC values in the low nanomolar range. The activity of the compounds was verified by binding experiments and supported by molecular modeling. The ability of the most effective inhibitors, used at non-toxic concentrations, to sensitize tumors to the anticancer drug topotecan was also demonstrated. The order of administration of the inhibitor and topotecan on their synergistic effect was studied, suggesting that prior or simultaneous introduction of the inhibitor with topotecan is the most effective.
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http://dx.doi.org/10.3390/molecules24203711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832265PMC
October 2019

DNA complexes with human apurinic/apyrimidinic endonuclease 1: structural insights revealed by pulsed dipolar EPR with orthogonal spin labeling.

Nucleic Acids Res 2019 09;47(15):7767-7780

N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, 9 Lavrentiev ave, Novosibirsk 630090, Russia.

A DNA molecule is under continuous influence of endogenous and exogenous damaging factors, which produce a variety of DNA lesions. Apurinic/apyrimidinic sites (abasic or AP sites) are among the most common DNA lesions. In this work, we applied pulse dipolar electron paramagnetic resonance (EPR) spectroscopy in combination with molecular dynamics (MD) simulations to investigate in-depth conformational changes in DNA containing an AP site and in a complex of this DNA with AP endonuclease 1 (APE1). For this purpose, triarylmethyl (TAM)-based spin labels were attached to the 5' ends of an oligonucleotide duplex, and nitroxide spin labels were introduced into APE1. In this way, we created a system that enabled monitoring the conformational changes of the main APE1 substrate by EPR. In addition, we were able to trace substrate-to-product transformation in this system. The use of different (orthogonal) spin labels in the enzyme and in the DNA substrate has a crucial advantage allowing for detailed investigation of local damage and conformational changes in AP-DNA alone and in its complex with APE1.
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http://dx.doi.org/10.1093/nar/gkz620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735896PMC
September 2019

Double-Stranded DNA Fragments Bearing Unrepairable Lesions and Their Internalization into Mouse Krebs-2 Carcinoma Cells.

Nucleic Acid Ther 2019 10 13;29(5):278-290. Epub 2019 Jun 13.

Laboratory of Induced Cell Processes, Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.

Murine Krebs-2 tumor-initiating stem cells are known to natively internalize extracellular double-stranded DNA fragments. Being internalized, these fragments interfere in the repair of chemically induced interstrand cross-links. In the current investigation, 756 bp polymerase chain reaction (PCR) product containing bulky photoreactive dC adduct was used as extracellular DNA. This adduct was shown to inhibit the cellular system of nucleotide excision repair while being resistant to excision by this DNA repair system. The basic parameters for this DNA probe internalization by the murine Krebs-2 tumor cells were characterized. Being incubated under regular conditions (60 min, 24°C, 500 μL of the incubation medium, in the dark), 0.35% ± 0.18% of the Krebs-2 ascites cells were shown to natively internalize modified DNA. The saturating amount of the modified DNA was detected to be 0.37 μg per 10 cells. For the similar unmodified DNA fragments, this ratio is 0.73 μg per 10 cells. Krebs-2 tumor cells were shown to be saturated internalizing either (190 ± 40) × 10 molecules of modified DNA or (1,000 ± 100) × 10 molecules of native DNA. On internalization, the fragments of DNA undergo partial and nonuniform hydrolysis of 3' ends followed by circularization. The degree of hydrolysis, assessed by sequencing of several clones with the insertion of specific PCR product, was 30-60 nucleotides.
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http://dx.doi.org/10.1089/nat.2019.0786DOI Listing
October 2019

A Single-Molecule Atomic Force Microscopy Study of PARP1 and PARP2 Recognition of Base Excision Repair DNA Intermediates.

J Mol Biol 2019 07 23;431(15):2655-2673. Epub 2019 May 23.

Institute of Chemical Biology and Fundamental Medicine (ICBFM) SB RAS, 8 Lavrentiev Avenue, Novosibirsk 630090, Russia. Electronic address:

Nuclear poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) catalyze the synthesis of poly(ADP-ribose) (PAR) and use NAD as a substrate for the polymer synthesis. Both PARP1 and PARP2 are involved in DNA damage response pathways and function as sensors of DNA breaks, including temporary single-strand breaks formed during DNA repair. Consistently, with a role in DNA repair, PARP activation requires its binding to a damaged DNA site, which initiates PAR synthesis. Here we use atomic force microscopy to characterize at the single-molecule level the interaction of PARP1 and PARP2 with long DNA substrates containing a single damage site and representing intermediates of the short-patch base excision repair (BER) pathway. We demonstrated that PARP1 has higher affinity for early intermediates of BER than PARP2, whereas both PARPs efficiently interact with the nick and may contribute to regulation of the final ligation step. The binding of a DNA repair intermediate by PARPs involved a PARP monomer or dimer depending on the type of DNA damage. PARP dimerization influences the affinity of these proteins to DNA and affects their enzymatic activity: the dimeric form is more effective in PAR synthesis in the case of PARP2 but is less effective in the case of PARP1. PARP2 suppresses PAR synthesis catalyzed by PARP1 after single-strand breaks formation. Our study suggests that the functions of PARP1 and PARP2 overlap in BER after a site cleavage and provides evidence for a role of PARP2 in the regulation of PARP1 activity.
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http://dx.doi.org/10.1016/j.jmb.2019.05.028DOI Listing
July 2019

Poly(ADP-ribosyl)ation and DNA repair synthesis in the extracts of naked mole rat, mouse, and human cells.

Aging (Albany NY) 2019 05;11(9):2852-2873

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

DNA repair capacity in cells of naked mole rat (Hgl), a species known for its longevity and resistance to cancer, is still poorly characterized. Here, using the whole-cell extracts (WCEs) of Hgl, mouse and human cells, we studied the interrelation between DNA synthesis on the substrates of base excision repair and the activity of poly(ADP-ribose) polymerases (PARPs) responsible for the transfer of the ADP-ribose moieties onto different targets. The level of PAR synthesis was more than ten-fold higher in human WCE as compared to rodent WCEs, while the efficiency of DNA synthesis was comparable. Under conditions of PAR synthesis, the efficiency of DNA synthesis was only slightly enhanced in all extracts and in mouse WCEs unusual products of the primer elongation were detected. The results obtained with WCEs, recombinant proteins and recently found ability of PARPs to attach the ADP-ribose moieties to DNA allowed us to attribute these products to primer mono(ADP-ribosyl)ation (MARylation) at the 5'-terminal phosphate by PARP3 during the DNA synthesis. PARP1/PARP2 can then transfer the ADP-ribose moieties onto initial ADP-ribose. Our results suggest that MARylation/PARylation of DNA in the extracts depends on the ratios between PARPs and can be controlled by DNA-binding proteins.
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http://dx.doi.org/10.18632/aging.101959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535076PMC
May 2019

PARP-1 Activation Directs FUS to DNA Damage Sites to Form PARG-Reversible Compartments Enriched in Damaged DNA.

Cell Rep 2019 05;27(6):1809-1821.e5

SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France. Electronic address:

PARP-1 synthesizes long poly(ADP-ribose) chains (PAR) at DNA damage sites to recruit DNA repair factors. Among proteins relocated on damaged DNA, the RNA-binding protein FUS is one of the most abundant, raising the issue about its involvement in DNA repair. Here, we reconstituted the PARP-1/PAR/DNA system in vitro and analyzed at the single-molecule level the role of FUS. We demonstrate successively the dissociation of FUS from mRNA, its recruitment at DNA damage sites through its binding to PAR, and the assembly of damaged DNA-rich compartments. PARG, an enzyme family that hydrolyzes PAR, is sufficient to dissociate damaged DNA-rich compartments in vitro and initiates the nucleocytoplasmic shuttling of FUS in cells. We anticipate that, consistent with previous models, FUS facilitates DNA repair through the transient compartmentalization of DNA damage sites. The nucleocytoplasmic shuttling of FUS after the PARG-mediated compartment dissociation may participate in the formation of cytoplasmic FUS aggregates.
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http://dx.doi.org/10.1016/j.celrep.2019.04.031DOI Listing
May 2019

Poly(ADP-ribosyl)ation by PARP1: reaction mechanism and regulatory proteins.

Nucleic Acids Res 2019 05;47(8):3811-3827

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

Poly(ADP-ribosyl)ation (PARylation) is posttranslational modification of proteins by linear or branched chains of ADP-ribose units, originating from NAD+. The central enzyme for PAR production in cells and the main target of poly(ADP-ribosyl)ation during DNA damage is poly(ADP-ribose) polymerase 1 (PARP1). PARP1 ability to function as a catalytic and acceptor protein simultaneously made a considerable contribution to accumulation of contradictory data. This topic is directly related to other questions, such as the stoichiometry of PARP1 molecules in auto-modification reaction, direction of the chain growth during PAR elongation and functional coupling of PARP1 with PARylation targets. Besides DNA damage necessary for the folding of catalytically active PARP1, other mechanisms appear to be required for the relevant intensity and specificity of PARylation reaction. Indeed, in recent years, PARP research has been enriched by the discovery of novel PARP1 interaction partners modulating its enzymatic activity. Understanding the details of PARP1 catalytic mechanism and its regulation is especially important in light of PARP-targeted therapy and may significantly aid to PARP inhibitors drug design. In this review we summarize old and up-to-date literature to clarify several points concerning PARylation mechanism and discuss different ways for regulation of PAR synthesis by accessory proteins reported thus far.
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http://dx.doi.org/10.1093/nar/gkz120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486540PMC
May 2019

Novel Inhibitors of DNA Repair Enzyme TDP1 Combining Monoterpenoid and Adamantane Fragments.

Anticancer Agents Med Chem 2019 ;19(4):463-472

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8, Lavrentiev Ave., Novosibirsk, 630090, Russian Federation.

Background And Objective: The DNA repair enzyme tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a current inhibition target to improve the efficacy of cancer chemotherapy. Previous studies showed that compounds combining adamantane and monoterpenoid fragments are active against TDP1 enzyme. This investigation is focused on the synthesis of monoterpenoid derived esters of 1-adamantane carboxylic acid as TDP1 inhibitors.

Methods: New esters were synthesized by the interaction between 1-adamantane carboxylic acid chloride and monoterpenoid alcohols. The esters were tested against TDP1 and its binding to the enzyme was modeling.

Results: 13 Novel ester-based TDP1 inhibitors were synthesized with yields of 21-94%; of these, nine esters had not been previously described. A number of the esters were found to inhibit TDP1, with IC50 values ranging from 0.86-4.08 µM. Molecular modelling against the TDP1 crystal structure showed a good fit of the active esters in the catalytic pocket, explaining their potency. A non-toxic dose of ester, containing a 3,7- dimethyloctanol fragment, was found to enhance the cytotoxic effect of topotecan, a clinically used anti-cancer drug, against the human lung adenocarcinoma cell line A549.

Conclusion: The esters synthesized were found to be active against TDP1 in the lower micromolar concentration range, with these findings being corroborated by molecular modeling. Simultaneous action of the ester synthesized from 3,7-dimethyloctanol-1 and topotecan revealed a synergistic effect.
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http://dx.doi.org/10.2174/1871520619666181207094243DOI Listing
February 2020

Dynamic light scattering study of base excision DNA repair proteins and their complexes.

Biochim Biophys Acta Proteins Proteom 2019 03 12;1867(3):297-305. Epub 2018 Oct 12.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia; Department of Natural Sciences, Novosibirsk State University, Novosibirsk 630090, Russia. Electronic address:

Base excision repair (BER) involves many enzymes acting in a coordinated fashion at the most common types of DNA damage. The coordination is facilitated by interactions between the enzymes and accessory proteins, X-ray repair cross-complementing protein 1 (XRCC1) and poly(ADP-ribose) polymerase 1 (PARP1). Here we use dynamic light scattering (DLS) technique to determine the hydrodynamic sizes of several BER enzymes and proteins, DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), tyrosyl-DNA phosphodiesterase 1 (TDP1), XRCC1 and PARP1, present alone or in the equimolar mixtures with each other. From the DLS data combined with glutaraldehyde cross-linking experiments and previous quantitative binding data the oligomeric states of BER proteins and their complexes are estimated. All the proteins have been proposed to form homodimers upon their self-association. The most probable oligomerization state of the binary complexes formed by PARP1 with various proteins is a heterotetramer. The oligomerization state of the binary complexes formed by XRCC1 varies from heterodimer to heterotetramer, depending on the partner. The DLS technique is applied for the first time to measure the hydrodynamic sizes of PARP1 molecules covalently bound with poly(ADP-ribose) (PAR) synthesized upon the automodification reaction. PARP1 has been detected to form huge conglomerates stabilized by Mg coordinated bonds with PAR polymers.
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http://dx.doi.org/10.1016/j.bbapap.2018.10.009DOI Listing
March 2019

Replication protein A as a modulator of the poly(ADP-ribose)polymerase 1 activity.

DNA Repair (Amst) 2018 12 24;72:28-38. Epub 2018 Sep 24.

Institute of Chemical Biology and Fundamental Medicine, Lavrentiev av. 8, Novosibirsk, 630090, Russia; Department of Natural Sciences, Novosibirsk State University, 1 Pirogov Street, Novosibirsk, 630090, Russia. Electronic address:

Replication protein A contributes to all major pathways of DNA metabolism and is a target for post-translation modifications, including poly(ADP-ribosyl)ation catalyzed by PARP1. Here we demonstrate that the efficiency of RPA poly(ADP-ribosyl)ation strongly depends on the structure of DNA used for PARP1 activation and on the polarity of RPA binding. Moreover, RPA influences PARP1 activity, and this effect also depends on DNA structure: RPA inhibits PAR synthesis catalyzed by PARP1 in the presence of ssDNA and stimulates it in the presence of a DNA duplex, in particular that containing a nick or a gap. Using fluorescently labeled proteins, we showed their direct interaction and characterized it quantitatively. RPA can accelerate the replacement of poly(ADP-ribosyl)ated PARP1 molecules bound to DNA by the unmodified ones. Thus, our data allow us to suggest that the balance between the affinities of PARP1 and RPA for DNA and the interaction of these proteins with each other are the cornerstone of the modulating effect of RPA on PARP1 activity. This effect might contribute to the regulation of PARP1 activity in various DNA processing mechanisms including DNA replication and repair pathways, where both PARP1 and RPA participate.
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http://dx.doi.org/10.1016/j.dnarep.2018.09.010DOI Listing
December 2018

A Novel Class of Tyrosyl-DNA Phosphodiesterase 1 Inhibitors That Contains the Octahydro-2-chromen-4-ol Scaffold.

Molecules 2018 Sep 26;23(10). Epub 2018 Sep 26.

Department of Natural Sciences and Institute of Medicine and Psychology, Novosibirsk State University, Pirogova 2, Novosibirsk 630090, Russia.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that mends topoisomerase 1-mediated DNA damage. Tdp1 is a current inhibition target for the development of improved anticancer treatments, as its inhibition may enhance the therapeutic effect of topoisomerase 1 poisons. Here, we report a study on the development of a novel class of Tdp1 inhibitors that is based on the octahydro-2-chromene scaffold. Inhibition and binding assays revealed that these compounds are potent inhibitors of Tdp1, with IC and values in the low micromolar concentration range. Molecular modelling predicted plausible conformations of the active ligands, blocking access to the enzymatic machinery of Tdp1. Our results thus help establish a structural-activity relationship for octahydro-2-chromene-based Tdp1 inhibitors, which will be useful for future Tdp1 inhibitor development work.
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http://dx.doi.org/10.3390/molecules23102468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222798PMC
September 2018

Novel group of tyrosyl-DNA-phosphodiesterase 1 inhibitors based on disaccharide nucleosides as drug prototypes for anti-cancer therapy.

J Enzyme Inhib Med Chem 2018 Dec;33(1):1415-1429

a Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences , Novosibirsk , Russian Federation.

A new class of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors based on disaccharide nucleosides was identified. TDP1 plays an essential role in the resistance of cancer cells to currently used antitumour drugs based on Top1 inhibitors such as topotecan and irinotecan. The most effective inhibitors investigated in this study have IC values (half-maximal inhibitory concentration) in 0.4-18.5 µM range and demonstrate relatively low own cytotoxicity along with significant synergistic effect in combination with anti-cancer drug topotecan. Moreover, kinetic parameters of the enzymatic reaction and fluorescence anisotropy were measured using different types of DNA-biosensors to give a sufficient insight into the mechanism of inhibitor's action.
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http://dx.doi.org/10.1080/14756366.2018.1509210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136360PMC
December 2018

In vitro lesion bypass by human PrimPol.

DNA Repair (Amst) 2018 10 27;70:18-24. Epub 2018 Jul 27.

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

Many human DNA polymerases bypass DNA damage during translesion synthesis (TLS). Human primase and polymerase, PrimPol, assists fork progression by repriming DNA synthesis downstream of the lesion using its DNA primase activity. We tested the properties of PrimPol as a TLS polymerase in the presence of different metal ions in vitro. We demonstrate that in the presence of Mg ions PrimPol carries out efficient and relatively accurate synthesis past 8-oxoguanine and 5-formyluracil. It also bypasses an abasic site and O-methylguanine, but is blocked by thymine glycol and 1,N-ethenoadenine. Substitution of Mg with Mn stimulates the TLS activity of PrimPol and allows for efficient, but error-prone, synthesis on DNA templates containing all tested DNA lesions, including thymine glycol and 1,N-ethenoadenine. The TLS activity of PrimPol has possible relevant functions in vivo; e.g., the combined primase and DNA polymerase activities of PrimPol might facilitate replication of DNA with clustered damage.
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http://dx.doi.org/10.1016/j.dnarep.2018.07.009DOI Listing
October 2018

The multifunctional protein YB-1 potentiates PARP1 activity and decreases the efficiency of PARP1 inhibitors.

Oncotarget 2018 May 4;9(34):23349-23365. Epub 2018 May 4.

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

Y-box-binding protein 1 (YB-1) is a multifunctional cellular factor overexpressed in tumors resistant to chemotherapy. An intrinsically disordered structure together with a high positive charge peculiar to YB-1 allows this protein to function in almost all cellular events related to nucleic acids including RNA, DNA and poly(ADP-ribose) (PAR). In the present study we show that YB-1 acts as a potent poly(ADP-ribose) polymerase 1 (PARP1) cofactor that can reduce the efficiency of PARP1 inhibitors. Similarly to that of histones or polyamines, stimulatory effect of YB-1 on the activity of PARP1 was significantly higher than the activator potential of Mg and was independent of the presence of EDTA. The C-terminal domain of YB-1 proved to be indispensable for PARP1 stimulation. We also found that functional interactions of YB-1 and PARP1 can be mediated and regulated by poly(ADP-ribose).
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http://dx.doi.org/10.18632/oncotarget.25158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955111PMC
May 2018

Unusual interaction of human apurinic/apyrimidinic endonuclease 1 (APE1) with abasic sites via the Schiff-base-dependent mechanism.

Biochimie 2018 Jul 3;150:88-99. Epub 2018 May 3.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia; Novosibirsk State University, Novosibirsk, Russia. Electronic address:

Clustered apurinic/apyrimidinic (AP) sites are more cytotoxic than isolated AP lesions because double strand breaks (DSB) can be formed during repair of closely positioned bistranded AP sites. Formation of DSB due to simultaneous cleavage of bistranded AP sites may be regulated by proteins specifically interacting with this complex lesion. A set of AP DNA duplexes containing AP sites in both strands in different mutual orientation (BS-AP DNAs) was used for search in the extracts of human cells proteins specifically recognizing clustered AP sites. A protein, which formed the Schiff-base-dependent covalent products having an apparent molecular mass of 50 kDa with the subset of BS-AP DNAs, was identified by mass spectrometry as apurinic/apyrimidinic endonuclease 1 (APE1). The identity of trapped protein was confirmed by Western blot analysis with anti-APE1 antibodies. Purified recombinant human APE1 is also capable of forming the 50 kDa-adducts with efficiency of BS-AP DNAs cross-linking to APE1 being dependent on the mutual orientation of AP sites. In spite of formation of the Schiff-base-dependent intermediate, which is prerequisite for the β-elimination mechanism, APE1 is unable to cleave AP sites. APE1 lacking the first 34 amino acids at the N-terminus, unlike wild type enzyme, is unable to form cross-links with BS-AP DNAs that testifies to the involvement of disordered N-terminal extension, which is enriched in lysine residues, in the interaction with AP sites. The yield of APE1-AP DNA cross-links was found to correlate with the enzyme amount in the extracts estimated by the immunochemical approach; therefore the BS-AP DNA-probes can be useful for comparative analysis of APE1 content in cell extracts.
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http://dx.doi.org/10.1016/j.biochi.2018.04.027DOI Listing
July 2018