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Effect of the M326V mutation on the relaxation and cleavage activities of YTOP. (A) YTOP-M326V has similar relaxation activity as wild-type YTOP enzyme. S: supercoiled plasmid DNA. (B) YTOP-M326V DNA cleavage activity was magnesium independent, and was enhanced over the wild-type DNA cleavage activity. Formation of oligonucleotide cleavage product was assayed with 400 ng of enzyme, 0.5 pmole of 5′-end labeled oligonucleotide substrate after incubation for 30 min at 37°C.
