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
.