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Photoactivated DNA analogs of substrates of the nucleotide excision repair system and their interaction with proteins of NER-competent HeLa cell extract

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Abstract

Photoactivated DNA analogs of nucleotide excision repair (NER) substrates have been created that are 48-mer duplexes containing in internal positions pyrimidine nucleotides with bulky substituents imitating lesions. Fluorochloroazidopyridyl, anthracenyl, and pyrenyl groups introduced using spacer fragments at 4N and 5C positions of dCMP and dUMP were used as model damages. The gel retardation and photo-induced affinity modification techniques were used to study the interaction of modified DNA duplexes with proteins in HeLa cell extracts containing the main components of NER protein complexes. It is shown that the extract proteins selectively bind and form covalent adducts with the model DNA. The efficiency and selectivity of protein modification depend on the structure of used DNA duplex. Apparent molecular masses of extract proteins, undergoing modification, were estimated. Mutual influence of simultaneous presence of extract proteins and recombinant NER protein factors XPC-HR23B, XPA, and RPA on interaction with the model DNA was analyzed. The extract proteins and RPA competed for interaction with photoactive DNA, mutually decreasing the yield of modification products. In this case the presence of extract proteins at particular concentrations tripled the increase in yield of covalent adducts formed by XPC. It is supposed that the XPC subunit interaction with DNA is stimulated by endogenous HR23B present in the extract. Most likely, the mutual effect of XPA and extract proteins stimulating formation of covalent adducts with model DNA is due to the interaction of XPA with endogenous RPA of the extract. A technique based on the use of specific antibodies revealed that RPA present in the extract is a modification target for photoactive DNA imitating NER substrates.

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Abbreviations

AB:

antibodies

Anthr-dCTP:

N-4-[4-(9-anthracenylhydrazinocarbonyl)butyl-carbamoyl]-2′-deoxycytidine-5′-triphosphate

Fap-dCTP:

exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl]aminoethyl}-2′-deoxycy-tidine-5′-triphosphate

HR23B:

58 kDa polypeptide, the small subunit of functional heterodimer XPC-HR23B

MBP-XPC:

XPC polypeptide containing an affinity cluster—maltose binding protein (MBP)

NER:

nucleotide excision repair

ONT:

oligonucleotides

Pyr-dUTP:

5-[N-(4-(1-pyrenyl)-ethylcarbonyl)-3-amino-trans-propenyl-1]-2′-deoxyuridine-5′-triphosphate

RPA:

human replication factor A

4S-dUTP:

4-thio-2′-deoxyuridine-5′-triphosphate

XPA and XPC:

Xeroderma pigmentosum factors of complementation groups A and C, respectively

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Authors and Affiliations

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

    I. O. Petruseva, E. A. Maltseva, I. V. Safronov & O. I. Lavrik

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

    I. S. Tikhanovich

Authors
  1. I. O. Petruseva
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  2. I. S. Tikhanovich
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  3. E. A. Maltseva
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  4. I. V. Safronov
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  5. O. I. Lavrik
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Correspondence to I. O. Petruseva.

Additional information

Original Russian Text © I. O. Petruseva, I. S. Tikhanovich, E. A. Maltseva, I. V. Safronov, O. I. Lavrik, 2009, published in Biokhimiya, 2009, Vol. 74, No. 5, pp. 607–619.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM08-290, February 15, 2009.

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Petruseva, I.O., Tikhanovich, I.S., Maltseva, E.A. et al. Photoactivated DNA analogs of substrates of the nucleotide excision repair system and their interaction with proteins of NER-competent HeLa cell extract. Biochemistry Moscow 74, 491–501 (2009). https://doi.org/10.1134/S0006297909050034

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  • DOI: https://doi.org/10.1134/S0006297909050034

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