Binding of the transcription factor, Sp1, to non-target sites in DNA modified by benzo[a]pyrene diol epoxide.
Covalent binding of the carcinogen, 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), to DNA causes changes in the conformation of the DNA around the site of the adduct. However, the influence of such carcinogen-DNA adducts on interactions of the DNA with specific proteins has received little attention. Binding of the transcription factor, Sp1, to GC-box sequences in the promoter of the hamster adenosine phosphoribosyl transferase gene is a useful model system. Electrophoretic mobility shift assays, competition experiments and DNase I footprinting demonstrated specific binding of affinity-purified, human Sp1 to two adjacent GC-boxes in the promoter fragment. Unexpectedly, modification of this DNA fragment to high levels (approximately 7% of the nucleotides) with BPDE caused a substantial (5- to 10-fold) increase in the apparent affinity of Sp1. A heterologous DNA fragment that contained no GC-boxes did not compete for the binding of Sp1 to the promoter, unless it was previously modified with BPDE. In addition, two DNA fragments that contained no GC-boxes exhibited Sp1-dependent mobility shifts only when modified by BPDE. DNase I footprinting of the BPDE-modified, Sp1-bound promoter fragment did not reveal specific sites of binding, suggesting that numerous BPDE-DNA adduct sites can interact with the protein. A model in which Sp1 binding to non-target sites is enhanced by a static bend or an induced flexibility at the site of an adduct is discussed.
MacLeod
M C
MC
Department of Carcinogenesis, University of Texas MD and Anderson Cancer Center, Smithville 78957, USA.
Powell
K L
KL
Tran
N
N
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D016384:Consensus Sequence; D018736:DNA Adducts / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000302:isolation & purification / Q000378:metabolism*
D018736:DNA Adducts; D009838:Oligodeoxyribonucleotides; D016329:Sp1 Transcription Factor; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D003850:Deoxyribonuclease I