Mutational analysis of the preferential binding of human topoisomerase I to supercoiled DNA.
Mutational analysis of the preferential binding of human topoisomerase I to supercoiled DNA.
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人拓扑异构酶 I 与超螺旋 DNA 优先结合的突变分析。
DOI：
10.1111/j.1742-4658.2009.07270.x
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Champoux,JamesJ
中科院分区：
文献类型：
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作者：
Yang,Zheng;Carey,JamesF;Champoux,JamesJ
人类拓扑异构酶I以拓扑依赖的方式结合DNA，其中对任一符号的超螺旋DNA的强烈偏好超过松弛环状DNA。 解释这种偏好的一个假设是，酶上存在第二个DNA结合位点，介导与超螺旋DNA中存在的节点的关联。即使在DNA存在下，酶也不能二聚化，这似乎排除了蛋白质二聚化产生两个结合位点的假设。产生了一系列突变蛋白构建体，以测试同源结构域样核心亚结构域II（残基233-319）提供第二个DNA结合位点，或者核心亚结构域III中的接头或碱性残基参与优先结合超螺旋DNA的假设。 当假定的核心亚结构域II内的DNA接触点被改变或该结构域被完全去除时，对酶识别超螺旋DNA的能力没有影响，如通过凝胶位移测定和竞争结合测定所测量的。 然而，对于缺乏卷曲螺旋接头区域的酶形式或当核心亚结构域III中的赖氨酸对变为谷氨酸时，对超螺旋的偏好明显降低。 所获得的结果表明，在人拓扑异构酶I与超螺旋DNA的优先结合中，核心亚结构域III中的接头和溶剂暴露的碱性残基。
Human topoisomerase I binds DNA in a topology‐dependent fashion with a strong preference for supercoiled DNAs of either sign over relaxed circular DNA. One hypothesis to account for this preference is that a second DNA‐binding site exists on the enzyme that mediates an association with the nodes present in supercoiled DNA. The failure of the enzyme to dimerize, even in the presence of DNA, appears to rule out the hypothesis that two binding sites are generated by dimerization of the protein. A series of mutant protein constructs was generated to test the hypotheses that the homeodomain‐like core subdomain II (residues 233–319) provides a second DNA‐binding site, or that the linker or basic residues in core subdomain III are involved in the preferential binding to supercoiled DNAs. When putative DNA contact points within core subdomain II were altered or the domain was removed altogether, there was no effect on the ability of the enzyme to recognize supercoiled DNA, as measured by both a gel shift assay and a competition binding assay. However, the preference for supercoils was noticeably reduced for a form of the enzyme lacking the coiled‐coil linker region or when pairs of lysines were changed to glutamic acids in core subdomain III. The results obtained implicate the linker and solvent‐exposed basic residues in core subdomain III in the preferential binding of human topoisomerase I to supercoiled DNA.