Publications by authors named "Irina O Petruseva"

8 Publications

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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

Structural basis for the recognition and processing of DNA containing bulky lesions by the mammalian nucleotide excision repair system.

DNA Repair (Amst) 2018 01 2;61:86-98. Epub 2017 Nov 2.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia; Novosibirsk State University, Ministry of Education and Science of the Russian Federation, Novosibirsk 630090, Russia; Altai State University, Ministry of Education and Science of the Russian Federation, Barnaul 656049, Russia. Electronic address:

Mammalian nucleotide excision repair (NER) eliminates the broadest diversity of bulky lesions from DNA with wide specificity. However, the double incision efficiency for structurally different adducts can vary over several orders of magnitude. Therefore, great attention is drawn to the question of the relationship among structural properties of bulky DNA lesions and the rate of damage elimination. This paper studies the properties of several structurally diverse synthetic (model) DNAs containing bulky modifications. Model DNAs have been designed using modified nucleosides (exo-N-{2-N-[N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl]aminoethyl}-2'-deoxycytidine (Fap-dC) and 5-{1-[6-(5[6]-fluoresceinylcarbomoyl)hexanoyl]-3-aminoallyl}-2'-deoxyuridine (Flu-dU)) and the nonnucleosidic reagent N-[6-(9-antracenylcarbomoyl)hexanoyl]-3-amino-1,2-propandiol (nAnt). The impact of these lesions on spatial organization and stability of the model DNA was evaluated. Their affinity for the damage sensor XPC was also studied. It was expected, that the values of melting temperature decrease, bending angles and K values clearly define the row of model DNA substrate properties such as Flu-dU-DNA>nAnt≈Fap-dC-DNA. Unexpectedly the experimentally estimated levels of the substrate properties were actually in the row: nAnt-DNA>Flu-dU-DNA>Fap-dC-DNA. Molecular dynamics simulations have revealed structural and energetic bases for the discrepancies observed. DNA destabilization patterns plotted explain these results on a structural basis in terms of differences in dynamic perturbations of stacking interactions.
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http://dx.doi.org/10.1016/j.dnarep.2017.10.010DOI Listing
January 2018

Comparative analysis of interaction of human and yeast DNA damage recognition complexes with damaged DNA in nucleotide excision repair.

J Biol Chem 2013 Apr 26;288(15):10936-47. Epub 2013 Feb 26.

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

The human XPC-RAD23B complex and its yeast ortholog, Rad4-Rad23, are the primary initiators of global genome nucleotide excision repair. The interaction of these proteins with damaged DNA was analyzed using model DNA duplexes containing a single fluorescein-substituted dUMP analog as a lesion. An electrophoretic mobility shift assay revealed similarity between human and yeast proteins in DNA binding. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed by fluorescent depolarization measurements. XPC-RAD23B and Rad4-Rad23 proteins demonstrate approximately equal binding affinity to the damaged DNA duplex (K(D) ∼ (0.5 ± 0.1) and (0.6 ± 0.3) nM, respectively). Using photoreactive DNA containing 5-iodo-dUMP in defined positions, XPC/Rad4 location on damaged DNA was shown. Under conditions of equimolar binding to DNA both proteins exhibited the highest level of cross-links to 5I-dUMP located exactly opposite the damaged nucleotide. The positioning of the XPC and Rad4 proteins on damaged DNA by photocross-linking footprinting is consistent with x-ray analysis of the Rad4-DNA crystal complex. The identity of the XPC and Rad4 location illustrates the common principles of structure organization of DNA damage-scanning proteins from different Eukarya organisms.
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http://dx.doi.org/10.1074/jbc.M112.444026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624473PMC
April 2013

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

Localization of xeroderma pigmentosum group A protein and replication protein A on damaged DNA in nucleotide excision repair.

Nucleic Acids Res 2010 Dec 6;38(22):8083-94. Epub 2010 Aug 6.

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

The interaction of xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA) with damaged DNA in nucleotide excision repair (NER) was studied using model dsDNA and bubble-DNA structure with 5-{3-[6-(carboxyamido-fluoresceinyl)amidocapromoyl]allyl}-dUMP lesions in one strand and containing photoreactive 5-iodo-dUMP residues in defined positions. Interactions of XPA and RPA with damaged and undamaged DNA strands were investigated by DNA-protein photocrosslinking and gel shift analysis. XPA showed two maximums of crosslinking intensities located on the 5'-side from a lesion. RPA mainly localized on undamaged strand of damaged DNA duplex and damaged bubble-DNA structure. These results presented for the first time the direct evidence for the localization of XPA in the 5'-side of the lesion and suggested the key role of XPA orientation in conjunction with RPA binding to undamaged strand for the positioning of the NER preincision complex. The findings supported the mechanism of loading of the heterodimer consisting of excision repair cross-complementing group 1 and xeroderma pigmentosum group F proteins by XPA on the 5'-side from the lesion before damaged strand incision. Importantly, the proper orientation of XPA and RPA in the stage of preincision was achieved in the absence of TFIIH and XPG.
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http://dx.doi.org/10.1093/nar/gkq649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001049PMC
December 2010

RPA repair recognition of DNA containing pyrimidines bearing bulky adducts.

J Mol Recognit 2008 May-Jun;21(3):154-62

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

Recognition of new DNA nucleotide excision repair (NER) substrate analogs, 48-mer ddsDNA (damaged double-stranded DNA), by human replication protein A (hRPA) has been analyzed using fluorescence spectroscopy and photoaffinity modification. The aim of the present work was to find quantitative characteristics of RPA-ddsDNA interaction and RPA subunits role in this process. The designed DNA structures bear bulky substituted pyrimidine nitrogen bases at the inner positions of duplex forming DNA chains. The photoreactive 4-azido-2,5-difluoro-3- pyridin-6-yl (FAP) and fluorescent antracenyl, pyrenyl (Antr, Pyr) groups were introduced via different linker fragments into exo-4N of deoxycytidine or 5C of deoxyuridine. J-dU-containing DNA was used as a photoactive model of undamaged DNA strands. The reporter group was a fluorescein residue, introduced into the 5'-phosphate end of one duplex-forming DNA strand. RPA-dsDNA association constants and the molar RPA/dsDNA ratio have been calculated based on fluorescence anisotropy measurements under conditions of a 1:1 RPA/dsDNA molar ratio in complexes. The evident preference for RPA binding to ddsDNA over undamaged dsDNA distinctly depends on the adduct type and varies in the following way: undamaged dsDNA < Antr-dC-ddsDNA < mmdsDNA < FAPdU-, Pyr-dU-ddsDNA < FAP-dC-ddsDNA (K(D) = 68 +/- 1; 25 +/- 6; 13 +/- 1; 8 +/- 2, and 3.5 +/- 0.5 nM correspondingly) but weakly depends on the chain integrity. Interestingly the bulkier lesions not in all cases have a greater effect on RPA affinity to ddsDNA. The experiments on photoaffinity modification demonstrated only p70 of compactly arranged RPA directly interacting with dsDNA. The formation of RPA-ddsDNA covalent adducts was drastically reduced when both strands of DNA duplex contained virtually opposite located FAP-dC and Antr-dC. Thus RPA requires undamaged DNA strand presence for the effective interaction with dsDNA bearing bulky damages and demonstrates the early NER factors characteristic features underlying strand discrimination capacity and poor activity of the NER system toward double damaged DNA.
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http://dx.doi.org/10.1002/jmr.877DOI Listing
October 2008

Crosslinking of nucleotide excision repair proteins with DNA containing photoreactive damages.

Bioorg Chem 2008 Apr 11;36(2):77-84. Epub 2008 Jan 11.

Laboratory of Bioorganic Chemistry of Enzymes, Institute of Chemical Biology and Fundamental Medicine, Lavrentiev Av. 8, 630090 Novosibirsk, Russia.

Photoreactive DNA duplexes mimicking substrates of nucleotide excision repair (NER) system were used to analyze the interaction of XPC-HR23B, RPA, and XPA with damaged DNA. Photoreactive groups in one strand of DNA duplex (arylazido-dCMP or 4-thio-dUMP) were combined with anthracenyl-dCMP residue at the opposite strand to analyze contacts of NER factors with damaged and undamaged strands. Crosslinking of XPC-HR23B complex with photoreactive 48-mers results in modification of XPC subunit. XPC-HR23B did not crosslink with DNA duplex bearing bulky residues in both strands while this modification does not prevent interaction of DNA with XPA. The data on crosslinking of XPA and RPA with photoreactive DNA duplexes containing bulky group in one of the strands are in favor of XPA preference to interact with the damaged strand and RPA preference for the undamaged strand. The results support the understanding and set the stage for dynamically oriented experiments of how the pre-incision complex is formed in the early stage of NER.
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http://dx.doi.org/10.1016/j.bioorg.2007.11.004DOI Listing
April 2008

Crosslinking of the NER damage recognition proteins XPC-HR23B, XPA and RPA to photoreactive probes that mimic DNA damages.

Biochim Biophys Acta 2007 May 25;1770(5):781-9. Epub 2007 Jan 25.

Institute of Chemical Biology and Fundamental Medicine, Lavrentiev av. 8, 630090 Novosibirsk, Russia.

A new assay to probe the mechanism of mammalian nucleotide excision repair (NER) was developed. Photoreactive arylazido analogues of dNMP in DNA were shown to be substrates for the human NER system. Oligonucleotides carrying photoreactive "damages" were prepared using the multi-stage protocol including one-nucleotide gap filling by DNA polymerase beta using photoreactive dCTP or dUTP analogues followed by ligation of the resulting nick. Photoreactive 60-mers were annealed with single-stranded pBluescript II SK (+) and subsequently primer extension reactions were performed. Incubation of HeLa extracts with the plasmids containing photoreactive moieties resulted in an excision pattern typical of NER. DNA duplexes containing photoreactive analogues were used to analyze the interaction of XPC-HR23B, RPA, and XPA with damaged DNA using the photocrosslinking assay. Crosslinking of the XPC-HR23B complex with photoreactive 60-mers resulted in modification of its XPC subunit. RPA crosslinked to ssDNA or mismatched dsDNA more efficiently than to dsDNA, whereas XPA did not show a preference for any of the DNA species. XPC and XPA photocrosslinking to DNA decreased in the presence of Mg(2+) whereas RPA crosslinking to DNA was not sensitive to this cofactor. Our data establish a photocrosslinking assay for the investigation of the damage recognition step in human nucleotide excision repair.
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http://dx.doi.org/10.1016/j.bbagen.2007.01.007DOI Listing
May 2007