Zhang H L, Malpure S, Li Z, Hiasa H, DiGate R J
Molecular and Cell Biology Program, University of Maryland, Baltimore, Maryland 21201, USA.
J Biol Chem. 1996 Apr 12;271(15):9039-45. doi: 10.1074/jbc.271.15.9039.
The role that the carboxyl-terminal amino acids of Escherichia coli DNA topoisomerase I (Topo I) and III (Topo III) play in catalysis was examined by comparing the properties of Topo III with those of a truncated enzyme lacking the generalized DNA binding domain of Topo III, Topo I, and a hybrid topoisomerase polypeptide containing the amino-terminal 605 amino acids of Topo III and the putative generalized DNA binding domain of Topo I. The deletion of the carboxyl-terminal 49 amino acids of Topo III decreases the affinity of the enzyme for its substrate, single-stranded DNA, by approximately 2 orders of magnitude and reduces Topo III-catalyzed relaxation of supercoiled DNA and Topo III-catalyzed resolution of DNA replication intermediates to a similar extent. Fusion of the carboxyl-terminal 312 amino acid residues of Topo I onto the truncated molecule stimulates topoisomerase-catalyzed relaxation 15-20-fold, to a level comparable with that of full-length Topo III. However, topoisomerase-catalyzed resolution of DNA replication intermediates was only stimulated 2-3-fold. Therefore, the carboxyl-terminal amino acids of these topoisomerases constitute a distinct and separable domain, and this domain is intimately involved in determining the catalytic properties of these polypeptides.
通过比较拓扑异构酶III（Topo III）与一种缺失Topo III的通用DNA结合结构域的截短酶、拓扑异构酶I（Topo I）以及一种包含Topo III的氨基末端605个氨基酸和Topo I假定的通用DNA结合结构域的杂合拓扑异构酶多肽的特性，研究了大肠杆菌DNA拓扑异构酶I（Topo I）和III（Topo III）的羧基末端氨基酸在催化中所起的作用。Topo III羧基末端49个氨基酸的缺失使其对底物单链DNA的亲和力降低了约2个数量级，并在相似程度上降低了Topo III催化的超螺旋DNA松弛以及Topo III催化的DNA复制中间体的拆分。将Topo I的羧基末端312个氨基酸残基融合到截短分子上可刺激拓扑异构酶催化的松弛反应15至20倍，达到与全长Topo III相当的水平。然而，拓扑异构酶催化的DNA复制中间体的拆分仅被刺激了2至3倍。因此，这些拓扑异构酶的羧基末端氨基酸构成一个独特且可分离的结构域，并且该结构域密切参与决定这些多肽的催化特性。
