Sequence-specific double-strand cleavage of DNA

In the presence of O2 and 5 mM dithiothreitol, penta-N-methylpyrrolecarboxamide-EDTA-Fe(II) [P5E*Fe(lI)] at 0.5 ILM cleaves pBR322 plasmid DNA (50 pAM in base pairs) on opposite strands to afford discrete DNA fragments as analyzed by agarose gel electrophoresis. High-resolution denaturing gel electrophoresis of a nP-end-labeled 517-base-pair restriction fragment containing a major cleavage site reveals that P5E-Fe(li) cleaves 3-5 base pairs contiguous to a 6-base-pair sequence, 5'-T-T-T-TT-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 5mM dithiothreitol, 0.01 pzM P5E-Fe(II) converts form I pBR322 DNA at 0.22 IzM 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, 25QC) at the 6-base-pair recognition level and may provide an approach to the design of "artificial restriction enzymes." 5' 5'