Abstract
The human dihydrofolate reductase (DHFR) promoter sequence contains two consensus binding sites for the Sp1 regulatory protein. We have determined the effect of intermolecular triplex DNA formation on Sp1 binding to the DHFR promoter. The DHFR Sp1 binding site I (-39 to -48 relative to the DHFR transcription start site) demonstrates concentration-dependent triplex formation with a 19-base pair G-rich oligonucleotide (GR19) which is complementary to the polypyrimidine strand. DNase I footprint analysis demonstrates that GR19 forms a DNA triplex structure with the DHFR promoter fragment in a sequence-specific manner. DNase I footprinting analysis also indicates that the orientation of binding of these G-rich oligonucleotides is antiparallel. CR19, a C-rich complementary oligonucleotide, on the other hand, does not form triplex. The DNase I protection pattern of DHFR promoter fragment incubated with both recombinant Sp1 and triplex-forming oligonucleotide suggests that triplex formation prevents Sp1 binding. This is confirmed by gel shift analysis which demonstrates that triplex formation by the Sp1 binding sequences of the DHFR promoter prevents recombinant Sp1 binding in a concentration-dependent manner. These results demonstrate that intermolecular triplex formation prevents regulatory protein binding in a sequence-specific manner.
Highlights
Dihydrofolate reductase is an important enzyme of folate moter sequence contains two consensus binding sites metabolism [7] which is expressed in all eukaryotic cells
We have demonstrated that the G-C specific DNA binding drug, mithramycin, which selectively inhibits DHFR expression, prevents Spl binding to the DHFR promoter
As shown inFig. 2A, gel retardation studies demonstrate that GR19 targeted to the Sbpinl ding site I of the DHFRpromoter forms triplex DNA when it is present in concentrations in excess of its complementary C-rich oligonucleotide
Summary
Vol 267,No 16,Issue of June 5, pp. 11163-11167,1992 Printed in U.S.A. Triplex Formation Prevents S p l Binding to the Dihydrofolate Reductase Promoter*. The human DHFR promoter sequence contains pendent triplex formationwith a 19-base pair G-rich two consensus binding sites for the Spl regulatory protein oligonucleotide (GR19) whicihs complementary to the [15]. The consensus Spl binding site is is confirmed bygel shift analysis which demonstrates not completely polypurine/polypyrimidine, these sequences that triplex formation by tShpel binding sequences of can form triple-stranded structures. The DHFR promoter is the DHFR promoterprevents recombinanSt pl binding ideal for these studies since it contains two Spl binding sites in aconcentration-dependentmanner.These results separated by 24bp with nonidentical flanking sequences. This demonstratethatintermolecular triplex formation provides an internal control to determine sequence specificity.
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