TY - JOUR

T1 - Purification and characterization of human topoisomerase I mutants

AU - Jensen, Anne Dam

AU - Svejstrup, Jesper Q.

PY - 1996

Y1 - 1996

N2 - 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 was 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 Tyr723Rhe, Arg488Gln and Lys532Glu in DNA relaxation and cleavage is not due to an inability of these mutants to bind DNA non-covalently.

AB - 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 was 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 Tyr723Rhe, Arg488Gln and Lys532Glu in DNA relaxation and cleavage is not due to an inability of these mutants to bind DNA non-covalently.

KW - DNA topoisomerase I

KW - mutagenesis

KW - nickel-affinity chromatography

U2 - 10.1111/j.1432-1033.1996.00389.x

DO - 10.1111/j.1432-1033.1996.00389.x

M3 - Journal article

C2 - 8612607

AN - SCOPUS:0030005883

VL - 236

SP - 389

EP - 394

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 2

ER -