Albany 2001

category image Biomolecular
SUNY at Albany
June 19-23, 2001

Sequence-specificity of p50 protein binding to ss and ds oligodeoxinucleotides investigated by means of biological microchip techniques.

P50 protein is the member of the Y-box binding transcription factor family (1,2). The generic oligonucleotide microchip was used to determine the sequence specificity of p50 binding to single- and double-stranded DNA. The generic microchip contained 4096 oligodeoxynucleotide 8-mers chemically immobilized within polyacrylamide gel pads on a glass slide (3). The two main approaches were used: binding of fluorescently labeled p50 to single-stranded oligonucleotides and p50 binding to oligonucleotide duplexes obtained by hybridization of generic microchip with a complementary mixture of fluorescently labeled 8-mer oligonucleotides. Oligodeoxynucleotide 8-mers and duplexes of about 10 b.p. were found to be long enough for analysis of p50 binding to ss and ds DNA forms. The study of p50 binding to all possible sequences of inner hexamers of oligonucleotide 8-mers made it possible to estimate that the most specific DNA sequence motives were G-rich ones. For the case of ds form p50 was found to destabilize double-stranded oligonucleotides while binding mainly to the one strand of each duplex. Motives for the most strongly destabilized complexes will be presented. The most specific to p50 protein sequences among all possible quadramer and pentamer motifs containing in the Y-box were determined.

References and Footnotes
  1. V.M. Evdokimova, A.S. Sitikov, P.N. Simonenko, O.A. Lazarev, K.S. Vasilenko, V.A. Ustinov, J.W.B. Hershey, L.P. Ovchinnikov, J. Biol. Chem. 270, 3186-3192 (1995).
  2. V.M. Evdokimova, L.P. Ovchinnikov, J. Biochem. Cell Bio. 31, 139-149 (1999).
  3. A.L. Drobyshev, A.S. Zasedatelev, G.M. Yershov, A.D. Mirzabekov, Nucleic Acids Res. 27, 4100-4105 (1999).

O.A. Zasedateleva (1)*, A.S. Krylov (1), D.V. Prokopenko (1), M.A. Skabkin (2), L.P. Ovchinnikov (2), A.D. Mirzabekov (1)

Engelhardt Institute of Molecular Biology of RAS(1) , 32 Vavilov Str., 119991, Moscow, Russia
Institute of Protein Research of RAS(2), Puschino, 142292, Moscow Region, Russia
*e-mail: for_lana@mail.ru