Das Benu Brata, Sen Nilkantha, Dasgupta Somdeb Bose, Ganguly Agneyo, Majumder Hemanta K
Department of Molecular Parasitology, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India.
J Biol Chem. 2005 Apr 22;280(16):16335-44. doi: 10.1074/jbc.M412417200. Epub 2005 Feb 12.
Leishmania donovani topoisomerase I is an unusual bisubunit enzyme. We have demonstrated earlier that the large and small subunit could be reconstituted in vitro to show topoisomerase I activity. We extend our biochemical study to evaluate the role of the large subunit in topoisomerase activity. The large subunit (LdTOP1L) shows a substantial degree of homology with the core DNA binding domain of the topoisomerase IB family. Two N-terminal truncation constructs, LdTOP1Delta39L (lacking amino acids 1-39) and LdTOP1Delta99L (lacking amino acids 1-99) of the large subunit were generated and mixed with intact small subunit (LdTOP1S). Our observations reveal that residues within amino acids 1-39 of the large subunit have significant roles in modulating topoisomerase I activity (i.e. in vitro DNA relaxation, camptothecin sensitivity, cleavage activity, and DNA binding affinity). Interestingly, the mutant LdTOP1Delta99LS was unable to show topoisomerase I activity. Investigation of the loss of activity indicates that LdTOP1Delta99L was unable to pull down glutathione S-transferase-LdTOP1S in an Ni(2+)-nitrilotriacetic acid co-immobilization experiment. For further analysis, we co-expressed LdTOP1L and LdTOP1S in Escherichia coli BL21(DE3)pLysS cells. The lysate shows topoisomerase I activity. Immunoprecipitation revealed that LdTOP1L could interact with LdTOP1S, indicating the subunit interaction in bacterial cells, whereas immunoprecipitation of bacterial lysate co-expressing LdTOP1Delta99L and LdTOP1S reveals that LdTOP1Delta99L was significantly deficient at interacting with LdTOP1S to reconstitute topoisomerase I activity. This study demonstrates that heterodimerization between the large and small subunits of the bisubunit enzyme appears to be an absolute requirement for topoisomerase activity. The residue within amino acids 1-39 from the N-terminal end of the large subunit regulates DNA topology during relaxation by controlling noncovalent DNA binding or by coordinating DNA contacts by other parts of the enzyme.
杜氏利什曼原虫拓扑异构酶I是一种独特的双亚基酶。我们之前已经证明，其大亚基和小亚基在体外可以重组以展现拓扑异构酶I活性。我们扩展了生化研究，以评估大亚基在拓扑异构酶活性中的作用。大亚基（LdTOP1L）与拓扑异构酶IB家族的核心DNA结合结构域具有高度同源性。构建了大亚基的两个N端截短体，即LdTOP1Delta39L（缺失氨基酸1 - 39）和LdTOP1Delta99L（缺失氨基酸1 - 99），并将它们与完整的小亚基（LdTOP1S）混合。我们的观察结果表明，大亚基氨基酸1 - 39内的残基在调节拓扑异构酶I活性（即体外DNA松弛、喜树碱敏感性、切割活性和DNA结合亲和力）方面具有重要作用。有趣的是，突变体LdTOP1Delta99LS无法展现拓扑异构酶I活性。对活性丧失的研究表明，在镍（2 +）-次氮基三乙酸共固定化实验中，LdTOP1Delta99L无法下拉谷胱甘肽S - 转移酶 - LdTOP1S。为了进一步分析，我们在大肠杆菌BL21(DE3)pLysS细胞中共表达LdTOP1L和LdTOP1S。裂解物展现出拓扑异构酶I活性。免疫沉淀显示LdTOP1L可以与LdTOP1S相互作用，表明在细菌细胞中存在亚基相互作用，而对共表达LdTOP1Delta99L和LdTOP1S的细菌裂解物进行免疫沉淀显示，LdTOP1Delta99L在与LdTOP1S相互作用以重组拓扑异构酶I活性方面存在显著缺陷。这项研究表明，双亚基酶的大亚基和小亚基之间的异源二聚化似乎是拓扑异构酶活性的绝对必要条件。大亚基N端氨基酸1 - 39内的残基通过控制非共价DNA结合或通过协调酶的其他部分与DNA的接触来调节DNA松弛过程中的DNA拓扑结构。
