Topoisomerase IV bends and overtwists DNA upon binding.
Escherichia coli topoisomerase IV (Topo IV) is an essential ATP-dependent enzyme that unlinks sister chromosomes during replication and efficiently removes positive but not negative supercoils. In this article, we investigate the binding properties of Topo IV onto DNA in the absence of ATP using a single molecule micromanipulation setup. We find that the enzyme binds cooperatively (Hill coefficient alpha approximately 4) with supercoiled DNA, suggesting that the Topo IV subunits assemble upon binding onto DNA. It interacts preferentially with (+) rather than (-) supercoiled DNA (Kd+=0.15 nM, Kd-=0.23 nM) and more than two orders-of-magnitude more weakly with relaxed DNA (Kd0 approximately 36 nM). Like gyrase but unlike the eukaryotic Topo II, Topo IV bends DNA with a radius 0= 6.4 nm and locally changes its twist and/or its writhe by 0.16 turn per bound complex. We estimate its free energy of binding and study the dynamics of interaction of Topo IV with DNA at the binding threshold. We find that the protein/DNA complex alternates between two states: a weakly bound state where it stays with probability p = 0.89 and a strongly bound state (with probability p = 0.11). The methodology introduced here to characterize the Topo IV/DNA complex is very general and could be used to study other DNA/protein complexes.
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Laboratoire de Physique Statistique, Ecole Normale Supérieure, UMR 8550 Centre National de la Recherche Scientifique, Paris, France. gcharvin@rockefeller.edu
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D000255:Adenosine Triphosphate / Q000737:chemistry; D001703:Biophysics / Q000379:methods; D004247:DNA / Q000737:chemistry*; D004261:DNA Replication; D027101:DNA Topoisomerase IV / Q000737:chemistry*; D004278:DNA, Superhelical / Q000737:chemistry; D004076:Digoxigenin / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D015233:Models, Statistical; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D013816:Thermodynamics; D013997:Time Factors
D004278:DNA, Superhelical; D000255:Adenosine Triphosphate; D004247:DNA; D027101:DNA Topoisomerase IV; D004076:Digoxigenin