title: Effects on NaeI-DNA recognition of the leucine to lysine substitution that transforms restriction endonuclease NaeI to a topoisomerase: a model for restriction endonuclease evolution.
abstract: Substituting lysine for leucine at position 43 (L43K) transforms NaeI from restriction endonuclease to topoisomerase and makes NaeI hypersensitive to intercalative anticancer drugs. Here we investigated DNA recognition by Nael-L43K. Using DNA competition and gel retardation assays, NaeI-L43K showed reduced affinity for DNA substrate and the ability to bind both single- and double-stranded DNA with a definite preference for the former. Sedimentation studies showed that under native conditions NaeI-L43K, like NaeI, is a dimer. Introduction of mismatched bases into double-stranded DNA significantly increased that DNA's ability to inhibit NaeI-L43K. Wild-type NaeI showed no detectable binding of either single-stranded DNA or mismatched DNA over the concentration range studied. These results demonstrate that the L43K substitution caused a significant change in recognition specificity by NaeI and imply that NaeI-L43K's topoisomerase activity is related to its ability to bind single-stranded and distorted regions in DNA. A mechanism is proposed for the evolution of the NaeI restriction-modification system from a topoisomerase/ligase by a mutation that abolished religation activity and provided a needed change in DNA recognition.
authors.affiliation: Lineberger Comprehensive Cancer Center and Department of Pathology, University of North Carolina Medical School, Chapel Hill 27599-7295, USA.
authors.forename: K
authors.initials: K
authors.lastname: Jo
authors.forename: M D
authors.initials: MD
authors.lastname: Topal
mesh_terms: D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D007930:Leucine / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008954:Models, Biological; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D013379:Substrate Specificity; D059004:Topoisomerase I Inhibitors
chemical_list: D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D059004:Topoisomerase I Inhibitors; D004247:DNA; C060630:endodeoxyribonuclease NaeI; D015252:Deoxyribonucleases, Type II Site-Specific; D004264:DNA Topoisomerases, Type I; D007930:Leucine; D008239:Lysine