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Biochemistry (Moscow)

, 74:491 | Cite as

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

  • I. O. PetrusevaEmail author
  • I. S. Tikhanovich
  • E. A. Maltseva
  • I. V. Safronov
  • O. I. Lavrik
Article

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.

Key words

DNA duplexes with bulky substituents protein factors of pre-incision NER complexes photoinduced affinity modification 

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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Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  • I. O. Petruseva
    • 1
    Email author
  • I. S. Tikhanovich
    • 2
  • E. A. Maltseva
    • 1
  • I. V. Safronov
    • 1
  • O. I. Lavrik
    • 1
  1. 1.Institute of Chemical Biology and Fundamental MedicineSiberian Branch of the Russian Academy of SciencesNovosibirskRussia
  2. 2.Novosibirsk State UniversityNovosibirskRussia

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