Sequence-specific double-strand cleavage of DNA by penta-N-methylpyrrolecarboxamide-EDTA·Fe(II)

Abstract

In the presence of O2 and 5 mM dithiothreitol, penta-N-methylpyrrolecarboxamide-EDTA·Fe(II) [P5E·Fe(II)] at 0.5 µ M cleaves pBR322 plasmid DNA (50 µ M 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·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 µ M P5E·Fe(II) converts form I pBR322 DNA at 0.22 µ M 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."