The effect of AT and GC sequence specific minor groove-binding agents on restriction endonuclease activity.
The ability of the naturally occurring A/T specific DNA minor groove binders netropsin and diastamycin A and two synthetic G/C selective oligopeptide analogues (1 and 2), to interfere with the catalytic activity of restriction endonucleases has been investigated. Enzymes were chosen to have A/T rich (EcoRI, EcoRV) or G/C rich (BalI, NruI) recognition sequences. An agarose gel assay was used to measure the cleavage of 32P-labelled DNA and ligand-DNA binding data was obtained using methidium-propyl EDTA footprinting. Netropsin and distamycin bind at the recognition sites, and dose-dependently inhibited cleavage by, EcoRI and EcoRV, (EcoRI > EcoRV). They were also more effective at inhibiting the catalytic activity of BalI than either 1 or 2. NruI was inhibited by distamycin and 2, but not by netropsin or 1. DNA footprinting revealed that neither 1 or 2 bound to the BalI or NruI recognition sequences under the conditions used whereas netropsin and distamycin footprint at adjacent sites. 1 binds to two of the three recognition sequences for the enzyme Fnu4HI (GCNGC) in the fragment studied and was shown to inhibit DNA cleavage only at these two sites. 2 binds strongly to two GGGCTC sequences which are recognition sites for the enzyme BanII. In this case a pronounced stimulation of cleavage was observed in the presence of 2 over a wide dose range. The results indicate that enzyme inhibition does not necessarily result from simultaneous occupancy of a common site, or at nearby flanking sequences, and in some circumstances, a pronounced stimulation of enzyme cleavage can occur.
Forrow
S M
SM
Department of Oncology, University College London Medical School, UK.
Lee
M
M
Souhami
R L
RL
Hartley
J A
JA
D001345:Autoradiography; D001482:Base Composition / Q000187:drug effects / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004214:Distamycins / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D007364:Intercalating Agents / Q000378:metabolism / Q000494:pharmacology*; D008024:Ligands; D008969:Molecular Sequence Data; D009429:Netropsin / Q000378:metabolism / Q000494:pharmacology*; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011485:Protein Binding / Q000187:drug effects; D013379:Substrate Specificity
D004214:Distamycins; D007364:Intercalating Agents; D008024:Ligands; D009842:Oligopeptides; D009429:Netropsin; C030000:stallimycin; D004247:DNA; D004262:DNA Restriction Enzymes