Biochemical analysis of the
DNA-binding properties of GL020924. (A) The chemical
structure of GL020924. (B) DNase I footprint of
GL020924 on DNA. Increasing concentrations of either netropsin or
GL020924 (amounts are indicated in nM above their respective lanes)
were assayed by DNase I footprinting as described in Materials and
Methods. The DNA sequence of the oligonucleotide is as described
in Materials and Methods and the footprinted region, TATTAATA, is
indicated by the arrow. (C) Various cyclin D1 promoter
sequences were used to compete with the fluorescent/DABCYL
indicator DNA duplex (5′-fluorescein-CTTTATTATTTT
and 3′-DABCYL-AAAATAATAAAG) for GL020924
binding. Competitor duplex DNAs were: wild-type cyclin D1 sequence,
5′-GGGAGTTTTGTTGAAGTTG-3′ (solid
circles); –30 to –21 mutant sequence, 5′-GGTCTGGGATCCGAAGTTG-3′ (open circles); 10 bp AT-rich site-containing sequence, 5′-GGGAGTTTTTTTTAAGTTG-3′ (solid triangles); 8 bp AT-rich site-containing sequence, 5′-GGGAGTTTTAAAAGAGTTG-3′ (open triangles); mutagenized bases are underlined, GL020924 concentration
was 1000 nM in all samples.