Photoactivated DNA analogs of substrates of the nucleotide excision repair system and their interaction with proteins of NER-competent extract of HeLa cells. Synthesis and application of long model DNA

Evdokimov, A.N.; Petruseva, I.O.; Pestryakov, P.E.; Lavrik, O.I.

Авторы: Evdokimov A.N., Petruseva I.O., Pestryakov P.E., Lavrik O.I.
Журнал: Biochemistry (Moscow)
Том: 76
Номер: 1
Год издания: 2011
Издательство: Pleiades Publishing, Ltd
Местоположение издательства: Road Town, United Kingdom
Первая страница: 157
Последняя страница: 166
DOI: 10.1134/s0006297911010159
Аннотация: Long linear DNA analogs of nucleotide excision repair (NER) substrates have been synthesized. They are 137-mer duplexes containing in their internal positions nucleotides with bulky substitutes imitating lesions with fluorochloroazidopyridyl and fluorescein groups introduced using spacer fragments at the 4N and 5C positions of dCMP and dUMP (Fap-dC- and Flu-dU-DNA) and DNA containing a (+)-cis-stereoisomer of benzo[a]pyrene-N2-deoxyguanidine (BP-dG-DNA, 131 bp). The interaction of the modified DNA duplexes with the proteins of NER-competent HeLa extract was investigated. The substrate properties of the model DNA in the reaction of specific excision were shown to vary in the series Fap-dC-DNA << Flu-dU-DNA < BP-dG-DNA. During the experiments on affinity modification of the proteins of NER-competent extract, Fap-dC-DNA (137 bp) containing a P-32-label in the photoactive nucleotide demonstrated properties of a highly efficient and selective probe. The set of the main targets of labeling included polypeptides of the extract with the same values of apparent molecular weights (35-90 kDa) as when using the shorter (48 bp) Fap-dC-DNA. Besides, some of the extract proteins were shown capable of specific and effective interaction with the long analog of NER substrate. Electrophoretic mobility of these proteins coincided with the mobilities of DNA-binding subunits of XPC-HR23B and PARP1 (similar to 127 and T]115 kDa, respectively). The 115-kDa target protein was identified as PARP1 using NAD(+)-based functional testing. The results suggest that the linear Fap-dC-DNA is an unrepairable substrate analog that can compete with effective NER substrates in the binding of the proteins responsible for lesion recognition and excision.
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Photoactivated DNA analogs of substrates of the nucleotide excision repair system and their interaction with proteins of NER-competent extract of HeLa cells. Synthesis and application of long model DNAстатья Перевод Исследовательская статья

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