Step-wise DNA relaxation and decatenation by NaeI-43K

Abstract

NaeI protein was originally isolated for its restriction endonuclease properties. NaeI was later discovered to either relax or cleave supercoiled DNA, depending upon whether NaeI position 43 contains a lysine (43K) or leucine (43L) respectively. NaeI-43K DNA relaxation activity appears to be the product of coupling separate endonuclease and ligase domains within the same polypeptide. Whereas NaeI relaxes supercoiled DNA like a topoisomerase, even forming a transient covalent intermediate with the substrate DNA, NaeI shows no obvious sequence similarity to the topoisomerases. To further characterize the topoisomerase activity of NaeI, we report here that NaeI-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 NaeI-43K. At low salt concentration NaeI-43K was processive; non-saturating amounts of enzyme relaxed a fraction of the DNA. At high salt concentration the same non-saturating amounts of NaeI-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, NaeI-43K decatenated kinetoplast DNA containing nicked circles, implying that NaeI-43K can cleave opposite a nick. The products of the reaction are decatenated nicked circles under both processive and distributive conditions. The behavior of NaeI-43K is consistent with that of a prokaryotic type I topoisomerase.