Study of Interaction of Human Replication Factor A with DNA Using New Photoreactive Analogs of dTTP

D. M. Kolpashchikov¹, P. E. Pestryakov¹, W. A. Wlassoff², S. N. Khodyreva¹, and O. I. Lavrik^1*

¹Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, pr. Akademika Lavrent'eva 8, Novosibirsk, 630090 Russia; fax: (383-2) 333-677; E-mail: lavrik@niboch.nsc.ru

²Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, pr. Akademika Lavrent'eva 10, Novosibirsk, 630090 Russia

^* To whom correspondence should be addressed.

Received August 19, 1999

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Replication factor A (RPA) is a protein that binds single-stranded DNA in eukaryotic cells; it participates in replication, repair, and recombination of DNA. RPA is composed of three subunits with molecular masses 70 (p70), 32 (p32), and 14 kD (p14). The photoaffinity labeling method was used to study the interaction of RPA with the 3´-end of duplex DNA containing extended 5´-end of a single strand. We have synthesized dTTP analogs containing photoreactive 2,3,5,6-tetrafluoro-4-azidobenzoyl group attached to the 5th position of the uracil residue with linkers of variable length (9, 11, and 13 atom chains). Using these analogs and dTTP analog containing the same photoreactive residue attached to the 5th position of the uracil residue with a 4-atom linker, a number of oligonucleotide primers carrying a single photoreactive group on the 3´-end were enzymatically synthesized. Using the complex of the photoreactive primers with DNA template containing extended 19-base 5´-end, human RPA was photoaffinity modified. The primers were covalently bound to the p70 and p32 subunits of RPA and the p14 subunit was not labeled by the primers. The data are discussed considering the previously suggested model of interaction of RPA with DNA during replication.

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KEY WORDS: photoaffinity modification, photoactivatable analogs of dNTP, replication protein A

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