DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme Nae I.
Sequence-specific DNA-protein interactions are basic to DNA function. To better understand these interactions, we studied the effect of position on cleavage of DNA by the type II restriction enzyme (EC 3.1.21.4) Nae I. We discovered two classes of Nae I restriction sites: sites susceptible and sites resistant to cleavage. Kinetic analysis showed that Nae I was activated by DNA containing cleavable Nae I sites to rapidly cleave resistant Nae I sites by a noncompetitive mechanism with a Km for substrate DNA of about 2 nM and a KA for activating DNA of about 6 nM; activation increased catalysis but not substrate binding. Deletion mutagenesis in vitro showed that sequences flanking the Nae I recognition site were responsible for the differences between activating and nonactivating Nae I sites. The polyamine spermidine had a dramatic effect on the interaction of Nae I with DNA; in the presence of 1 mM spermidine, resistant sites were cleaved rapidly and cleavable DNA inhibited cleavage. The direct regulation of enzymatic activity by DNA sequences in trans, and the modulation of this regulation by a polyamine that is sensitive to the cell cycle, provides a regulatory switch mechanism. The implications of this switch for biological control functions are discussed.
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Lineberger Cancer Research Center, University of North Carolina Medical School, Chapel Hill 27599-7295.
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D001483:Base Sequence; D002872:Chromosome Deletion; D004247:DNA; D004269:DNA, Bacterial; D004279:DNA, Viral; D015252:Deoxyribonucleases, Type II Site-Specific / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010957:Plasmids; D013095:Spermidine / Q000494:pharmacology*; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D004247:DNA; C060630:endodeoxyribonuclease NaeI; D015252:Deoxyribonucleases, Type II Site-Specific; D013095:Spermidine