Purification and characterization of human topoisomerase I mutants.
A system for rapid purification and characterization of eukaryotic topoisomerase-I mutants has been developed. The system utilizes six-histidine tagging of human topoisomerase I expressed in Saccharomyces cerevisiae to enable purification by nickel-affinity chromatography. Virtually homogenous mutant proteins are then tested for their ability to relax supercoiled DNA plasmids and their capacity for binding, cleaving and religating short defined DNA substrates. Relaxation-deficient mutants were obtained by site-directed mutagenesis of selected highly conserved amino acids. The mutants Tyr723Phe (active site mutation), Arg488Gln and Lys532Glu were inert in relaxation of DNA, whereas Lys720Glu showed a 50-fold reduction in specific relaxation activity. Accordingly, only Lys720Glu showed low, but detectable cleavage activity on suicide DNA substrates, uncoupling the cleavage and religation events of topoisomerase I. The relative religation efficiency of Lys720Glu comparable to that of wild-type topoisomerase I, indicating that Lys720 is involved in interactions important for normal DNA cleavage, but not for the religation reaction. All mutants could be cross linked by ultraviolet light to bromo-dUTP-substituted DNA oligonucleotides carrying a topoisomerase-I-binding site, indicating that the deficiency of Tyr723Phe, Arg488Gln and Lys532Glu in DNA relaxation and cleavage is not due to an inability of these mutants to bind DNA non-covalently.
Jensen
A D
AD
Department of Molecular Biology, University of Aarhus, Denmark.
Svejstrup
J Q
JQ
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics
D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004264:DNA Topoisomerases, Type I