Preferential binding of human topoisomerase I to superhelical DNA (original) (raw)

Abstract

Eukaryotic type I DNA topoisomerase provides swivels for removing torsional strain from the DNA helix during transcription and DNA replication. Previously it has been shown that the enzyme is associated with actively transcribed genes and replicating DNA. Using an inactive mutant form of the protein containing phenylalanine instead of tyrosine at position 723, we have investigated the binding properties of the protein as a function of substrate topology. A series of filter binding assays indicated that the protein strongly prefers to bind superhelical over completely relaxed SV40 DNA. The ability of a supercoiled DNA to compete against a relaxed DNA for binding increases as the number of superhelical turns in the DNA increases. Since positively supercoiled DNA is bound with the same preference as negatively supercoiled DNA, we hypothesize that topoisomerase I binds preferentially at the nodes created 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 the C-terminus. These results suggest that this core domain may target the enzyme in vivo to regions of torsionally strained superhelical DNA.

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