Sequence-specific double-strand cleavage of DNA by penta-N-methylpyrrolecarboxamide-EDTA X Fe(II).
In the presence of O2 and 5 mM dithiothreitol, penta-N-methylpyrrolecarboxamide-EDTA X Fe(II) [P5E X Fe(II)] at 0.5 microM cleaves pBR322 plasmid DNA (50 microM in base pairs) on opposite strands to afford discrete DNA fragments as analyzed by agarose gel electrophoresis. High-resolution denaturing gel electrophoresis of a 32P-end-labeled 517-base-pair restriction fragment containing a major cleavage site reveals that P5E X Fe(II) cleaves 3-5 base pairs contiguous to a 6-base-pair sequence, 5'-T-T-T-T-T-A-3' (4,323-4,328 base pairs). The major binding orientation of the pentapeptide occurs with the amino terminus at the adenine side of this sequence. In the presence of 5 mM dithiothreitol, 0.01 microM P5E X Fe(II) converts form I pBR322 DNA at 0.22 microM plasmid (1.0 mM in base pairs) to 40% form II, indicating the cleavage reaction is catalytic, turning over a minimum of nine times. This synthetic molecule achieves double-strand cleavage of DNA (pH 7.9, 25 degrees C) at the 6-base-pair recognition level and may provide an approach to the design of "artificial restriction enzymes".
D001483:Base Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D004269:DNA, Bacterial; D004229:Dithiothreitol; D004492:Edetic Acid / Q000031:analogs & derivatives*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011758:Pyrroles
D004269:DNA, Bacterial; D011758:Pyrroles; C039806:penta-N-methylpyrrolecarboxamide-EDTA-Fe(II); D004492:Edetic Acid; D004229:Dithiothreitol