Effects of 8-oxo-7,8-dihydro-2 '-deoxyguanosine on the binding of the transcription factor Sp1 to its cognate target DNA sequence (GC box)

Emphasis was placed in this work on the assessment of the role of guanine b ases in the interaction of transcription factor Sp1 with its cognate DNA se quence. For this purpose, each guanine residue of the 5'-GGGGCGGGG-3' (GC b ox) target DNA sequence was substituted in turn by 8-oxo-7,8-dihydro-2'-deo xyguanosine. The latter oxidized nucleotide which is likely to be present i n mammalian DNA and exhibit mutogenic features is expected to be involved i n age-related diseases and cancer. The effect of the incorporation of 8-oxo dGuo into DNA on the binding of transcription factor Sp1 was studied using electrophoretic mobility shift assays with nuclear extracts from HeLa cells . When guanines at position G'2, G'3, G'4, G'5 and G'6 were replaced with 8 -oxodGuo, binding of Sp1 was only 28%, 30%, 7%, 5% and 21%, respectively, t o that of the non-substituted oligonucleotide. The binding is less affected when guanines at position G'1, G'7, G'8 and G'9 were substituted by 8-oxod Guo. Results show up the importance of the core of the GC box and the stron ger contribution of the second and the third zinc finger to the binding wit h DNA. All together, this suggests that incorporation of 8-oxodGuo may alte r the expression of the gene regulated by Sp1 and affect the response of th e cell.