Step-wise DNA relaxation and decatenation by NaeI-43K.
Nae I protein was originally isolated for its restriction endonuclease properties. Nae I was later discovered to either relax or cleave supercoiled DNA, depending upon whether Nae I position 43 contains a lysine (43K) or leucine (43L) respectively. Nae I-43K DNA relaxation activity appears to be the product of coupling separate endonuclease and ligase domains within the same polypeptide. Whereas Nae I relaxes supercoiled DNA like a topoisomerase, even forming a transient covalent intermediate with the substrate DNA, Nae I shows no obvious sequence similarity to the topoisomerases. To further characterize the topoisomerase activity of Nae I, we report here that Nae I-43K changes the linking number of a single negatively supercoiled topoisomer of pBR322 by units of one and therefore is a type I topoisomerase. Positively supercoiled pBR322 was resistant to Nae I-43K. At low salt concentration Nae I-43K was processive; non-saturating amounts of enzyme relaxed a fraction of the DNA. At high salt concentration the same non-saturating amounts of Nae I-43K partially relaxed all the DNA in a step-wise fashion to give a Gaussian distribution of topoisomers, demonstrating a switch from a processive to a distributive mode of action. Nae I-43K decatenated kinetoplast DNA containing nicked circles, implying that Nae I-43K can cleave opposite a nick. The products of the reaction are decatenated nicked circles under both processive and distributive conditions. The behavior of Nae I-43K is consistent with that of a prokaryotic type I topoisomerase.
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Lineberger Comprehensive Cancer Center and Department of Pathology, University of North Carolina Medical School, Chapel Hill, NC 27599-7295, USA.
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D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry*; D018105:DNA, Kinetoplast / Q000737:chemistry; D004278:DNA, Superhelical / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D008239:Lysine / Q000502:physiology; D015636:Magnesium Chloride / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology
D004269:DNA, Bacterial; D018105:DNA, Kinetoplast; D004278:DNA, Superhelical; D015636:Magnesium Chloride; D012965:Sodium Chloride; C060630:endodeoxyribonuclease NaeI; D015252:Deoxyribonucleases, Type II Site-Specific; D004264:DNA Topoisomerases, Type I; D008239:Lysine