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 topoisomerase and recombinase (NaeI-L43K) that shows no sequence similarity to these protein families. This transformation appears to result from coupled endonuclease and ligase domains. To further elucidate the relationship between NaeI-L43K and the topoisomerase protein family, we studied the effect of the topoisomerase inhibitors on NaeI-L43K activity. 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 NaeI and NaeI-L43K were compared. The drugs hardly inhibited DNA cleavage 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 intercalative drugs, except for ethidium bromide, enhance production of topoisomerase--DNA covalent intermediates but inhibited production of the NaeI-L43K--DNA covalent intermediate. These results imply some unique differences between DNA relaxation by NaeI-L43K and DNA topoisomerase. Concomitant with studying inhibition of the cleavage intermediate, NaeI-L43K was found to covalently bond with the 5' end of the cleaved DNA strand.
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Lineberger Comprehensive Cancer Center, University of North Carolina Medical School, Chapel Hill 27599-7295, USA.
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D000677:Amsacrine / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D003630:Daunorubicin / Q000494:pharmacology; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000378:metabolism*; D004611:Ellipticines / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019426:Integrases; D007364:Intercalating Agents / Q000494:pharmacology*; D007700:Kinetics; D008239:Lysine; D009268:Nalidixic Acid / Q000494:pharmacology; D009675:Novobiocin / Q000494:pharmacology; D010093:Oxolinic Acid / Q000494:pharmacology; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D045522:Recombinases; D059004:Topoisomerase I Inhibitors
D000970:Antineoplastic Agents; D004611:Ellipticines; D004791:Enzyme Inhibitors; D007364:Intercalating Agents; D011994:Recombinant Proteins; D045522:Recombinases; D059004:Topoisomerase I Inhibitors; D000677:Amsacrine; C034192:ellipticine; D009675:Novobiocin; D009268:Nalidixic Acid; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C115006:integron integrase IntI1; D015246:Deoxyribonuclease EcoRI; C060630:endodeoxyribonuclease NaeI; D015252:Deoxyribonucleases, Type II Site-Specific; D004264:DNA Topoisomerases, Type I; D008239:Lysine; D010093:Oxolinic Acid; D003630:Daunorubicin