Crosslinking of SRF to the c-fos SRE CArG box guanines using photo-active thioguanine oligodeoxynucleotides.
6-Thioguanine (thioG) was chemically incorporated into 25-base oligodeoxynucleotides encoding the c-fos serum response element (SRE) at positions corresponding to each guanine of the CArG box, which only slightly impaired DNA binding by the Serum Response Factor (SRF). Upon exposure to long wavelength UV light each thioG-containing SRE could be crosslinked to SRF, with efficiencies ranging from < 1 to 25% of the complex depending on the position of thioG in the SRE and on the UV source used. Crosslinking was strongest to the 3' side of the CArG box, and to the outer rather than the inner CArG box guanines, consistent with hydrogen bonds formed between SRF and the outer guanines in the crystal structure [Pellegrini et al., Nature 376, 490, 1995]. The crosslinked product was found to be chemically unstable. Possible mechanisms of crosslink formation are discussed.
Cahill
M A
MA
Hannover Medical School, Institute for Molecular Biology, Germany.
Nordheim
A
A
Xu
Y Z
YZ
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D016762:Genes, fos; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000737:chemistry; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D026362:Serum Response Factor; D013866:Thioguanine / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D026362:Serum Response Factor; D014157:Transcription Factors; D013866:Thioguanine