Figure 1.

DNA cleavage by EcoKMcrA. The reactions were performed with 0.5 μM wild type EcoKMcrA (dimer) and 0.2 μM of unmodified, hemi- or fully methylated oligoduplex DNA or single stranded DNA, at 37°C in a buffer supplemented with 0.1 mM Mn²⁺. Top strand and bottom strand cleavage positions on the gels are marked by blue and red dotted lines, respectively. Gel lanes ‘S’ contained radiolabeled single-stranded oligonucleotides corresponding to the 5′-terminal fragments of the respective DNA strands (sizes in nucleotides are shown on the sides of the gels). The amounts of the bottom and top strand cleavage products after 3 hours of digestion are plotted as blue/red arrows along the oligoduplex sequences. The uncleaved oligonucleotides (the 0 hour lanes) contained a detectable fraction (∼10%) of shorter fragments. The reported cleavage data was corrected for the amount of these contaminants.