Isolation and Partial Characterisation of the Relaxation Protein from Nuclei of Cultured Mouse and Human Cells

Abstract

A protein, called relaxation protein because of its ability to remove superhelical turns in closedcircular DNA, has been isolated and partially characterized from the nuclei of LA9 mouse and HeLa cells. The purification was facilitated by an assay method, with PM2 DNA, which used the fluorescence enhancement of the intercalating dye ethidium bromide upon binding to the closedcircular DNA. The amount of dye bound depends upon the degree of the superhelix density of the DNA. The relaxation products were analysed by the buoyant separation method in CsCl containing ethidium bromide and were shown to be completely relaxed. The purification resulted in a single band in a dodecylsulfate gel electrophoresis with an apparent molecular weight of 37000. The pH optimum is 7.0 and the optimal salt concentration is 0.2 M NaC1. The relaxation protein removes negative as well as positive supercoils, the latter generated by the interaction of ethidium bromide with closed-circular DNA. Relaxation of positive supercoils results, after removal of the dye, in the formation of molecules with superhelix densities exceeding that of native PM2 DNA (0.054). The highest negative superhelix density observed was -0.098 k 0.001. The corresponding positive superhelix density has been calculated to be + 0.023.A nicking -swivelling -closing mechanism is postulated, but nicked intermediates have so far not been demonstrated. The relaxation protein is not inhibited by known mammalian endonuclease I inhibitors, except for denatured DNA, and does not possess a conventional polynucleotide ligase activity. The relaxation activity was found to be predominantly in the nuclei, with only small amounts present in the cytoplasm and mitochondria. The biological function of transient swivels induced by the relaxation protein is not known. However, transient swivels are considered necessary or useful in the replication of closed-circular DNA or long linear DNA, respectively. Relaxation protein could replace the combined action of an endonuclease and a ligase ahead of the replication fork. Alternatively, transient swivels could be involved in the transcription process.In 1971 Wang [l] isolated and extensively purified a protein called o from uninfected Escherichiu coli cells. This protein was able to remove supercoils from negatively supercoiled DNA by reducing the topological winding number, a process which requires that the polynucleotide chain be nicked and then be rejoined after swivelling has occurred. Since the reaction did not occur with positively supercoiled DNA, it seemed doubtful that the co protein could replace the combined actions of an endonuclease and a polynucleotide ligase at the growing fork of duplex DNA during DNA replication. Such an action would reduce the length of duplex DNA that rotates aheadEnzymes. E. coli alkaline phosphatase (EC 3.1.3.1); pancreatic DNase (EC 3.1.4.5). of the growing fork during semi-conservative replication.Champoux and Dulbecco [2] in 1972, found a similar relaxation activity in sonicated nuclei of embryonic ...

Analysis of Covalent Complexes Formed between Calf Thymus DNA Topisomerase and Single-Stranded DNA

“…The specific radioactivities of the nucleotides at the time of purchase were 3000 Ci/mmol and 382 Ci/mmol respectively. Buffer A is 20 mM NaCI, 10 mM Tris-HCI, pH 7 (BcrmH1) and T4 polynucleotide kinase were from Boehringer (Mannheim). Proteinase K was from Merck (Darmstadt).…”

DNA-relaxing enzyme from Micrococcus luteus

“…In preliminary experiments we found that the M.luteus enzyme was inactive on positively twisted PM2 DNAs (generated by addition of ethidium bromide) under conditions under which the DNA-relaxing enzyme from human fibroblasts (Witte and Thielmann, unpublished) did relax these substrates. Quite in contrast to the eukaryotic enzymes (2)(3)(4)6,12), the w protein from E.coli is also inactive on positively twisted superhelical DNA substrates (1,5).…”

“…9b). DISCUSSION DNA-relaxing enzymes were first found in E.coli (1), subsequently in various eukaryotic cells (2,4,6,12,13) and recently in vaccinia virus (11).…”

“…The enzymes may be involved in any process requiring winding or unwinding of the double helix such as replication (8) or transcription (9). DNA-relaxing activities have been purified from Escherichia coli (1,10), vaccinia virus (11), Drosophila melanogaster (3), yeast (Thielmann and Hess, in preparation), calf thymus (12), rat liver (13), mouse embryo cells (4), HeLa (4) and KB cells (6). The enzymes of eukaryotic origin are distinguished from the w protein of E.coli in that they relaxe both negatively and positively twisted DNAs and they do not require Mg++ for activity (1)(2)(3)(4)6,12,13).…”

“…DNA-relaxing enzymes catalyze the conversion of superhelical DNA to a non-superhelical covalently closed form (1)(2)(3)(4) presumably by introducing a transient swivel into the helix (5). The relaxation process probably does not follow a single-hit mechanism as intermediates are generated having smaller numbers of superhelical turns than the original DNA substrate (3,5,6).…”