EFFECTS ON NAEI-DNA RECOGNITION OF THE LEUCINE TO LYSINE SUBSTITUTIONTHAT TRANSFORMS RESTRICTION-ENDONUCLEASE NAEI TO A TOPOISOMERASE - A MODEL FOR RESTRICTION-ENDONUCLEASE EVOLUTION

Substituting lysine for leucine at position 43 (L43K) transforms Nael from restriction endonuclease to topoisomerase and makes Nael hypersen sitive to intercalative anticancer drugs. Here we investigated DNA rec ognition by Nael-L43K. Using DNA competition and gel retardation assay s, Nael-L43K showed reduced affinity for DNA substrate and the ability to bind both single- and double-stranded DNA with a definite preferen ce for the former. Sedimentation studies showed that under native cond itions Nael-L43K, like Nael, is a dimer. Introduction of mismatched ba ses into double-stranded DNA significantly increased that DNA's abilit y to inhibit Nael-L43K. Wild-type Nael showed no detectable binding of either single-stranded DNA or mismatched DNA over the concentration r ange studied. These results demonstrate that the L43K substitution cau sed a significant change in recognition specificity by Nad and imply t hat Nael-L43K's topoisomerase activity is related to its ability to bi nd single-stranded and distorted regions in DNA, A mechanism is propos ed for the evolution of the Nael restriction-modification system from a topoisomerase/ligase by a mutation that abolished religation activit y and provided a needed change in DNA recognition.