EXPRESSION OF YEAST DNA TOPOISOMERASE-I CAN COMPLEMENT A CONDITIONAL-LETHAL DNA TOPOISOMERASE-I MUTATION IN ESCHERICHIA-COLI
EXPRESSION OF YEAST DNA TOPOISOMERASE-I CAN COMPLEMENT A CONDITIONAL-LETHAL DNA TOPOISOMERASE-I MUTATION IN ESCHERICHIA-COLI
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DOI：
10.1073/pnas.84.24.8971
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发表时间：
1987-12-01
影响因子：
11.1
通讯作者：
WANG, JC
中科院分区：
文献类型：
--
作者：
BJORNSTI, MA;WANG, JC
我们发现，尽管在一级结构，底物偏好和催化机制的差异，酵母DNA拓扑异构酶I可以在功能上取代大肠杆菌DNA拓扑异构酶I。利用表达酵母TOP 1基因或其5“缺失突变体的质粒家族来补充E. colitopA突变体。然后通过快速裂解程序从细胞中分离这些质粒，并检查它们的超螺旋化程度。在topA中的一个条件致死突变的功能互补，它编码E。大肠杆菌DNA拓扑异构酶I，与催化活性酵母酶的表达相关，该酶降低细胞内DNA的负超螺旋程度。我们还表明，. apprxeq。该酶的氨基端部分的130个氨基酸可以被删除而不影响其体外活性;然而，大肠杆菌对这种缺失更敏感。
We show that, despite differences in primary structure, substrate preference, and mechanism of catalysis, yeast DNA topoisomerase I can functionally substitute for Escherichia coli DNA topoisomerase I. A family of plasmids expressing the yeast TOP1 gene or 5''-deletion mutations of it were used to complement the temperature-sensitive phenotype of an E. coli topA mutant. These plasmids were then isolated from the cells by a rapid lysis procedure and examined for their degrees of supercoiling. Functional complementation of a conditional-lethal mutation in topA, which encodes E. coli DNA topoisomerase I, correlates with the expression of a catalytically active yeast enzyme that reduces the degree of negative supercoiling of intracellular DNA. We also show that .apprxeq. 130 amino acids of the amino-terminal portion of the yeast enzyme can be deleted without affecting its activity in vitro; activity of the enzyme inside E. coli, however, is more sensitive to such deletions.