The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange
The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange
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DOI：
10.1074/jbc.m117.791392
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发表时间：
2017-08-04
影响因子：
4.8
通讯作者：
Johnson, Kenneth A.
中科院分区：
文献类型：
--
作者：
Qian, Yufeng;Johnson, Kenneth A.
人线粒体ssDNA结合蛋白（mtSSB）是一种同源四聚体蛋白，参与线粒体DNA的复制和维持。虽然mtSSB在结构上类似于大肠杆菌的SSB（EcoSSB），但它缺乏C-末端无序结构域，并且对mtSSB-ssDNA相互作用的生物物理学知之甚少。在这里，我们的特点是mtSSB结合ssDNA的平衡滴定和停流动力学测量的动力学和热力学。我们发现mtSSB四聚体可以以两种不同的结合模式与ssDNA结合：（SSB）（30）和（SSB）（60），分别由30和60个核苷酸的DNA结合位点大小定义。我们发现，结合模式是由镁离子和NaCl浓度调制，但不像EcoSSB，mtSSB不显示负亚基间协同。平衡和动力学数据的全局拟合提供了控制（SSB）（60）和（SSB）（30）复合物形成的速率和平衡常数以及两种结合模式之间的转变的估计值。我们发现mtSSB四聚体与ssDNA结合的速率常数接近扩散极限（2 × 10（9）m（-1）s（-1）），较长的DNA（60个核苷酸）迅速包裹所有四个单体，如FRET分析所示。我们还表明，mtSSB四聚体可以直接从一个ssDNA分子转移到另一个通过中间体与两个DNA分子结合的mtSSB。总之，我们的研究结果表明，人类mtSSB与EcoSSB共享许多物理化学特性，并且这些差异可以通过人类mtSSB蛋白中缺乏酸性、无序的C-末端尾来解释。
The human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB-ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB)(30) and (SSB)(60), defined by DNA binding site sizes of 30 and 60 nucleotides, respectively. We found that the binding mode is modulated by magnesium ion and NaCl concentration, but unlike EcoSSB, the mtSSB does not show negative intersubunit cooperativity. Global fitting of both the equilibrium and kinetic data afforded estimates for the rate and equilibrium constants governing the formation of (SSB)(60) and (SSB)(30) complexes and for the transitions between the two binding modes. We found that the mtSSB tetramer binds to ssDNA with a rate constant near the diffusion limit (2 x 10(9) m(-1) s(-1)) and that longer DNA (60 nucleotides) rapidly wraps around all four monomers, as revealed by FRET assays. We also show that the mtSSB tetramer can directly transfer from one ssDNA molecule to another via an intermediate with two DNA molecules bound to the mtSSB. In conclusion, our results indicate that human mtSSB shares many physicochemical properties with EcoSSB and that the differences may be explained by the lack of an acidic, disordered C-terminal tail in human mtSSB protein.