Targeting of the HIV-1 long terminal repeat with chromomycin potentiates the inhibitory effects of a triplex-forming oligonucleotide on Sp1–DNA interactions and in vitro transcription

Abstract

We have studied the effects of chromomycin and of a triple-helix-forming oligonucleotide (TFO) that recognizes Sp1 binding sites on protein-DNA interactions and HIV-1 transcription. Molecular interactions between chromomycin, the Sp1 TFO and target DNA sequences were studied by gel retardation, triplex affinity capture using streptavidin-coated magnetic beads and biosensor technology. We also determined whether chromomycin and a TFO recognizing the Sp1 binding sites of the HIV-1 long terminal repeat (LTR) inhibit the activity of restriction enzyme HaeIII, which recognizes a sequence (5'-GGCC-3') located within these Sp1 binding sites. The effects of chromomycin and the TFO on the interaction between nuclear proteins or purified Sp1 and a double-stranded oligonucleotide containing the Sp1 binding sites of the HIV-1 LTR were studied by gel retardation. The effects of both chromomycin and TFO on transcription were studied by using an HIV-1 LTR-directed in vitro transcription system. Our results indicate that low concentrations of chromomycin potentiate the effects of the Sp1 TFO in inhibiting protein-DNA interactions and HIV-1-LTR-directed transcription. In addition, low concentrations of chromomycin do not affect binding of the TFO to target DNA molecules. The results presented here support the hypothesis that both DNA binding drugs and TFOs can be considered as sequence-selective modifiers of DNA-protein interactions, possibly leading to specific alterations of biological functions. In particular, the combined use of chromomycin and TFOs recognizing Sp1 binding sites could be employed in order to abolish the biological functions of promoters (such as the HIV-1 LTR) whose activity is potentiated by interactions with the promoter-specific transcription factor Sp1.

Interaction of the Human NF-κB p52 Transcription Factor with DNA-PNA Hybrids Mimicking the NF-κB Binding Sites of the Human Immunodeficiency Virus Type 1 Promoter

“…The sensor chip SA5 (research grade; Amersham Pharmacia Biotech) precoated with streptavidin was used. All procedures were performed at 25°C and at a 5 l/min flow rate (42,43). The production of SA5 sensor chips containing NF-B target double-stranded DNA-DNA (42), DNA-PNA, and PNA-PNA molecules was achieved by (a) a 30-l pulse (500 ng) injection of biotinylated NF-B oligomer (5Ј-TGGG-GACTTTCCAG-3Ј), either biot(NF-B)DNA or biot(NF-B)PNA, to two different flow cells of the sensor chip, followed by (b) a 30-l injection (500 ng) of the complementary NF-B oligomer (5Ј-CTGGAAAGTC-CCCA-3Ј), either c(NF-B)DNA or c(NF-B)PNA, as required ( Fig.…”

“…1 shows the location of NF-B binding sites within the HIV-1 LTR). Generation of double-stranded NF-B molecules was performed in HBS buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.05% surfactant P2) in order to minimize triple-helix formation (43).…”

Deoxysugar Transfer during Chromomycin A 3 Biosynthesis in Streptomyces griseus subsp. griseus : New Derivatives with Antitumor Activity

“…In this respect, they have been shown to prevent resistance to other antitumor agents by a number of mechanisms, including downregulation of proteins, such as MDR1 (16,28). Chromomycin and the closely related compound mithramycin were also found to stimulate K562 cell erythroid differentiation (3), to be potent inhibitors of neuronal apoptosis (7), and to have antiviral activity against human immunodeficiency virus type 1 (2).…”

Comparative Analyses of Gene Clusters and Ks-alpha Genes Involved in the Biosynthesis of Chromomycin A3 and Mithramycin

“…These polyketides have properties of inhibiting growth and multiplication of many tumor cell lines [2] . Chromomycin A 3 and mithramycin have been shown to have a stimulatory effect on K562 cell erythroid differentiation [3] and have also been found as neurological therapeutics [4] and in treatment of HIV-1 [5] .…”