Accessibility of nuclear chromatin by DNA binding polyamides.
Pyrrole-imidazole polyamides bind DNA with affinities comparable to those of transcriptional regulatory proteins and inhibit the DNA binding activities of components of the transcription apparatus. If polyamides are to be useful for the regulation of gene expression in cell culture experiments, one pivotal issue is accessibility of specific sites in nuclear chromatin. We first determined the kinetics of uptake and subcellular distribution of polyamides in lymphoid and myeloid cells using fluorescent polyamide-bodipy conjugates and deconvolution microscopy. Then cells were incubated with a polyamide-chlorambucil conjugate, and the sites of specific DNA cleavage in the nuclear chromatin were assayed by ligation-mediated PCR. In addition, DNA microarray analysis revealed that two different polyamides generated distinct transcription profiles. Remarkably, the polyamides affected only a limited number of genes.
Dudouet
Brigitte
B
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Burnett
Ryan
R
Dickinson
Liliane A
LA
Wood
Malcolm R
MR
Melander
Christian
C
Belitsky
Jason M
JM
Edelson
Benjamin
B
Wurtz
Nicholas
N
Briehn
Christoph
C
Dervan
Peter B
PB
Gottesfeld
Joel M
JM
D000478:Alkylation; D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D020869:Gene Expression Profiling; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism; D008856:Microscopy, Fluorescence; D022423:Myeloid Cells / Q000166:cytology / Q000378:metabolism; D009757:Nylons / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D020411:Oligonucleotide Array Sequence Analysis; D014158:Transcription, Genetic / Q000187:drug effects*
D002843:Chromatin; D009757:Nylons; D004247:DNA