Functional domains of Escherichia coli single-stranded DNA binding protein as assessed by analyses of the deletion mutants.
Functional domains of Escherichia coli single-stranded DNA binding protein as assessed by analyses of the deletion mutants.
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通过分析缺失突变体评估大肠杆菌单链 DNA 结合蛋白的功能域。
DOI：
10.1021/bi961647s
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发表时间：
1997
期刊：
影响因子：
2.9
通讯作者：
M. Shimizu
中科院分区：
文献类型：
--
作者：
T. Kinebuchi;H. Shindo;H. Nagai;N. Shimamoto;M. Shimizu
构建了一系列大肠杆菌单链DNA结合蛋白（SSB）的C-和N-末端缺失突变体，纯化并表征了自身多聚化和结合DNA的能力。高效凝胶过滤色谱分析表明，89-105位氨基酸在177个氨基酸的天然SSB的同源四聚体的维持中起关键作用。有趣的是，这里研究的所有N-末端缺失突变体都以八聚体形式洗脱，表明N-末端11个残基参与阻止八聚体的形成。用凝胶迁移率变动分析和圆二色性光谱研究了SSB及其缺失突变体蛋白与单链d（T）n的结合。C-末端缺失突变体蛋白，SSB 1 -135和SSB 1 -115，保持高亲和力，并可以以与天然SSB相同的方式被单链DNA（ssDNA）包裹。相反，C-末端区域（残基89-115）或N-末端区域（残基1-11）的缺失引起结合亲和力的显著降低。此外，对于天然SSB、SSB 1 -135、SSB 1 - 115和SSB 37 -177，观察到SSB在与d（T）64而不是与d（T）32的复合物中的两种不同化学计量，这表明（SSB）65和（SSB）35结合模式，如先前所证明的[Lohman，T. M.，& Overman，L. B。（1985）J.Biol.Chem.260，3594-3603; Bujalowski，W.，& Lohman，T. M.（1986）Biochemistry 25，7799-7802]，分别在较低和较高SSB浓度下发生。给出了SSB分子的功能图并进行了讨论。
A series of C- and N-terminal deletion mutants of Escherichia coli single-stranded DNA binding protein (SSB) was constructed, purified, and characterized in terms of ability to self-multimerize and to bind to DNA. High-performance gel filtration chromatography revealed that the amino acids 89-105 play a key role in the maintenance of homotetramer for native SSB of 177 amino acids. Interestingly, all of the N-terminal deletion mutants studied here were eluted as octamers, indicating that the N-terminal 11 residues are involved in the prevention of the formation of octamers. The binding of SSB and its deletion mutant proteins to single-stranded d(T)n was examined by gel mobility shift assay and circular dichroism spectroscopy. C-terminal deletion mutant proteins, SSB1-135 and SSB1-115, maintained high affinity and may be wrapped by single-stranded DNA (ssDNA) in the same way as in the case of native SSB. In contrast, deletion of the C-terminal region (residues 89-115) or N-terminal region (residues 1-11) caused a dramatic decrease in the binding affinity. Furthermore, two different stoichiometries of SSB in the complexes with d(T)64, but not with d(T)32, were observed for native SSB, SSB1-135, SSB1-115, and SSB37-177, suggesting that the (SSB)65 and (SSB)35 binding modes, as previously demonstrated [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799-7802], occurred at lower and higher SSB concentrations, respectively. A functional map for SSB molecule was presented and discussed.