Liu Q, Wang J C
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
J Biol Chem. 1998 Aug 7;273(32):20252-60. doi: 10.1074/jbc.273.32.20252.
Site-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands. Complementation tests show that alanine substitution at Arg-690, Asp-697, Lys-700, Arg-704, or Arg-781, but not at His-735, His-736, Glu-738, Gln-750, or Asn-828, inactivates the enzyme in vivo. Measurements of DNA relaxation and cleavage by purified mutant enzymes show that these activities are abolished in the R690A mutant and are much reduced in the mutants D697A, K700A, R704A, and R781A. When a Y782F polypeptide with a phenylalanine substituting for the active site tyrosine was expressed in cells that also express the R690A polypeptide, the resulting heterodimeric yeast DNA topoisomerase II was found to nick plasmid DNA. Thus in a dimeric wild-type enzyme, Tyr-782 in one protomer and Arg-690 in the other cooperate in trans in the catalysis of DNA cleavage. For the residues D697A, K700A, R704A, and R781A, their locations in the crystal structures of type II DNA topoisomerase fragments suggest that Arg-781 and Lys-700 might be involved in anchoring the 5' and 3' sides of the broken DNA, respectively, and the roles of Asp-697 and Arg-704 are probably less direct.
对酵母DNA拓扑异构酶II C端一半区域内的10个高度保守的极性残基进行了定点诱变，该区域对应于细菌DNA促旋酶的A亚基，以确定在DNA链的断裂和重新连接过程中增强活性位点酪氨酸Tyr-782作用的氨基酸侧链。互补试验表明，在Arg-690、Asp-697、Lys-700、Arg-704或Arg-781处进行丙氨酸替代会使酶在体内失活，但在His-735、His-736、Glu-73,8、Gln-750或Asn-828处进行丙氨酸替代则不会。对纯化的突变酶进行DNA松弛和切割测量表明，这些活性在R690A突变体中被消除，而在D697A、K700A、R704A和R781A突变体中则大大降低。当在也表达R690A多肽的细胞中表达用苯丙氨酸替代活性位点酪氨酸的Y782F多肽时，发现产生的异源二聚体酵母DNA拓扑异构酶II会切割质粒DNA。因此，在二聚体野生型酶中，一个亚基中的Tyr-782和另一个亚基中的Arg-690在DNA切割催化中进行反式协作。对于D697A、K700A、R704A和R781A残基，它们在II型DNA拓扑异构酶片段晶体结构中的位置表明，Arg-781和Lys-700可能分别参与固定断裂DNA的5'和3'端，而Asp-697和Arg-704的作用可能不太直接。
