CHANGING A LEUCINE TO A LYSINE RESIDUE MAKES NAEI ENDONUCLEASE HYPERSENSITIVE TO DNA INTERCALATIVE DRUGS

A Single amino acid change transforms restriction enzyme NaeI to a top oisomerase and recombinase (NaeI-L43K) that shows no sequence similari ty to these protein families. This transformation appears to result fr om coupled endonuclease and ligase domains. To further elucidate the r elationship between NaeI-L43K and the topoisomerase protein family, we studied the effect of the topoisomerase inhibitors on NaeI-L43K activ ity. The intercalative drugs amsacrine, ellipticine, and daunorubicin inhibited NaeI-L43K, whereas the nonintercalating drugs camptothecin, VP-16, and oxolinic acid did not. Ethidium bromide also inhibited NaeI -L43K, Implying that intercalation is responsible for its inhibition. The effects of the intercalative drugs on the DNA cleavage steps of Na eI and NaeI-L43K were compared. The drugs hardly inhibited DNA cleavag e by wild type NaeI but completely inhibited DNA cleavage by NaeI-L43K . This difference in inhibition demonstrates that the L43K amino acid change sensitized NaeI to these drugs. Low concentrations of the inter calative drugs, except for ethidium bromide, enhance production of top oisomerase-DNA covalent intermediates but inhibited production of the NaeI-L43K-DNA covalent intermediate. These results imply some unique d ifferences between DNA relaxation by NaeI-L43K and DNA topoisomerase. Concomitant with studying inhibition of the cleavage intermediate, Nae I-L43K was found to covalently bond with the 5' end of the cleaved DNA strand.