Use of double-stranded DNA mini-circles to characterize the covalent topoisomerase-DNA complex

Abstract

The enzymatic DNA relaxation requires the DNA to be transiently nicked and rejoined, the covalent topoisomerase-DNA complex being a key intermediate of the nicking-joining reaction. Practically, this reaction is most often characterized by oligonucleotides. However, the incision-religation of an oligonucleotide does not fully recapitulate the incision-religation occuring during relaxation and the preferred substrate for such reaction characterization is supercoiled DNA. We therefore developed a method that used radiolabeled supercoiled DNA mini-circles to characterize the covalent enzyme-DNA complex formed during a relaxation reaction. Resolution of the relaxation products under different conditions permitted to quantify the proportion of covalent complex formed during the relaxation catalyzed by two topoisomerase models, the Escherichia coli topoisomerase I and the calf thymus topoisomerase I. As expected, the covalent complex formed with the calf thymus topoisomerase I was significantly enriched by camptothecin, a widely-used inhibitor of this topoisomerase, and a salt jump permitted the multiple topoisomerases trapped per mini-circle to complete the reaction cycle. The identified positions of the camptothecin-induced incision sites were shown to be independent of the linking number and the substrate circular nature Overall, our results demonstrate that supercoiled mini-circles constitute a powerful and polyvalent substrate to characterize the mechanism of action of novel topoisomerases and inhibitors, including the incision-religation reaction.

Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins

“…The results show that the products of the “in gel” relaxation reaction are T -1 in case of the treatment of the complex C a or C b with the Ec TopoI and T 0/-1 in case of the treatment of the complex C a or C b with the wheat germ Topoisomerase ( Fig 8B ). Knowing that Ec TopoI has no activity on T -1 topoisomers and that the wheat germ Topoisomerase relaxes T -1 topoisomers into T 0 topoisomers [ 28 ], our results indicate that the binding of hRPA on T -1 topoisomer does not induce a topological change in the dsMC.…”

“…The procedure to prepare dsMCs of 235 base pairs with a specific linking number has been published elsewhere [ 28 ] and is presented on the S1 Fig . Briefly, the dephosphorylated DNA fragment of 235 base pairs was radiolabeled with T4 polynucleotide kinase [2 μM [gamma - 32 P]-ATP, 0.6 U microL -1 PNK, 30 min, 37°C in polynucleotide kinase buffer (70 mM Tris-HCl pH 7.6, 10 mM MgCl 2 , 5 mM DTT)] and ligated with T4 DNA ligase (2 U microL -1 , 4 h, 16°C in T4 DNA ligase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 1 mM ATP, 10 mM DTT) in the presence of various concentrations of ethidium bromide (EtBr, from 0 to 20 microg mL -1 ).…”

“…For both types of experiments, the DNA substrates used were double-stranded DNA mini-circles (dsMCs) for several reasons (reviewed in [ 27 ]). First, several methods (including our method [ 28 ]) of preparation of dsMCs make it possible to produce topoisomers whose topology ranges from relaxed to hyper-negatively or positively supercoiled forms of DNA [ 25 , 29 – 31 ]. Second, dsMCs have proved to be suitable substrates to characterize the biochemical activity of topoisomerases [ 30 , 32 , 33 ] and the structural properties of DNA, either naked [ 17 , 18 , 23 , 25 , 26 , 34 ] or assembled into chromatin [ 29 , 32 , 35 – 37 ].…”