Mutational analysis of Escherichia coli topoisomerase IV. I. Selection of dominant-negative parE alleles.
In order to define regions of ParE, one of the two subunits of topoisomerase IV, that are involved in catalysis during topoisomerization, we developed a selection procedure to isolate dominant-negative parE alleles. Both wild-type parC and mutagenized parE were expressed from a tightly-regulated lac promoter on a moderate-copy plasmid. Mutated parE alleles were rescued from those plasmids that caused IPTG-dependent cell death. The mutant ParE proteins could be divided into two groups when reconstituted with ParC to form topoisomerase IV, those that elicited hyper-DNA cleavage and those that affected covalent complex formation.
Mossessova
E
E
Molecular Biology Graduate Program, Cornell University Graduate School of Medical Sciences, New York, New York 10021, USA.
Levine
C
C
Peng
H
H
Nurse
P
P
Bahng
S
S
Marians
K J
KJ
D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics*; D027101:DNA Topoisomerase IV; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004278:DNA, Superhelical / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D010641:Phenotype
D001426:Bacterial Proteins; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D007544:Isopropyl Thiogalactoside; D027101:DNA Topoisomerase IV; D004250:DNA Topoisomerases, Type II