PREFERENTIAL BINDING OF HUMAN TOPOISOMERASE-I TO SUPERHELICAL DNA

Eukaryotic type I DNA topoisomerase provides swivels for removing tors ional strain from the DNA helix during transcription and DNA replicati on. Previously it has been shown that the enzyme is associated with ac tively transcribed genes and replicating DNA. Using an inactive mutant form of the protein containing phenylalanine instead of tyrosine at p osition 723, we have investigated the binding properties of the protei n as a function of substrate topology, A series of filter binding assa ys indicated that the protein strongly prefers to bind superhelical ov er completely relaxed SV40 DNA. The ability of a supercoiled DNA to co mpete against a relaxed DNA for binding increases as the number of sup erhelical turns in the DNA increases. Since positively supercoiled DNA is bound with the same preference as negatively supercoiled DNA, we h ypothesize that topoisomerase I binds preferentially at the nodes crea ted by the crossing of two duplex helices. The preference for binding superhelical DNA is also exhibited by the conserved core domain (amino acids 175-659) which is missing the active site region located near t he C-terminus. These results suggest that this core domain may target the enzyme in vivo to regions of torsionally strained superhelical DNA .