Mutations of vaccinia virus DNA topoisomerase I that stabilize the cleavage complex.
Two mutations in vaccinia virus topoisomerase I, K167D and G226N, have been characterized. SOS induction was observed in Escherichia coli expressing vaccinia topoisomerase I with either one of these mutations. The mutant enzymes were purified to homogeneity and compared with the wild type enzyme for relaxation activity and the partial activities of substrate binding, site-specific DNA cleavage and DNA religation to determine the mechanism of SOS induction. The K167D mutant enzyme had reduced binding affinity for the DNA substrate with a Kapp that was 10-fold higher than wild type. Nevertheless, in reactions with high enzyme concentration, its substrate cleavage activity was 90% that of wild type. The G226N mutant enzyme had virtually wild type binding and cleavage activities. However, intermolecular religation by these two mutants were observed to be significantly reduced. The cleavage complexes formed with the K167D and G226N mutants were more stable to high salt than the wild type cleavable complex. We propose that these mutants in vivo induce the SOS response in E. coli due to the shift of topoisomerase cleavage-religation equilibrium towards cleavage and increased stability of the cleavage complex. The mutation thus has a similar effect as the topoisomerase-targeting inhibitors that turn topoisomerases into DNA damaging agents.
Gupta
M
M
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595.
Zhu
C X
CX
Tse-Dinh
Y C
YC
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010957:Plasmids; D013014:SOS Response, Genetics; D012965:Sodium Chloride / Q000494:pharmacology; D014616:Vaccinia virus / Q000201:enzymology*
D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D012965:Sodium Chloride; D004247:DNA; D004264:DNA Topoisomerases, Type I