Mechanism of Interaction between Single-Stranded DNA Binding Protein and DNA
Mechanism of Interaction between Single-Stranded DNA Binding Protein and DNA
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DOI：
10.1021/bi901743k
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发表时间：
2010-02-09
期刊：
影响因子：
2.9
通讯作者：
Webb, Martin R.
中科院分区：
文献类型：
--
作者：
Kunzelmann, Simone;Morris, Caroline;Webb, Martin R.
用荧光团标记的单链 DNA 结合蛋白 (SSB) 与单链 DNA (ssDNA) 相互作用，使荧光增强 6 倍。标记的蛋白质是来自大肠杆菌的同源四聚体SSB的G26C突变体和二乙氨基香豆素{N-[2-(碘乙酰胺)乙基]-7-二乙氨基香豆素-3-甲酰胺}的加合物。该加合物可用于测定解旋酶分离双链 DNA 过程中 ssDNA 的产生。为了有效地使用该探针，以及研究 ssDNA 和 SSB 之间的相互作用，荧光 SSB 已被用来开发蛋白质和 ssDNA 结合和解离的动力学机制。在类似于 70 个碱基长度的 ssDNA 包裹在四聚体周围的条件下，初始缔合相对简单且快速，可能是扩散控制的。 70 个碱基长度的 ssDNA（结合一个四聚体）和聚（dT）（可以结合多个四聚体）的动力学相似。在某些条件下（高SSB和/或低离子强度），第二个四聚体与每个70个碱基长度结合，但结合速率比第一个四聚体的结合速率慢2个数量级。解离动力学很复杂，并且由于游离野生型 SSB 的存在而大大加速。荧光 SSB.ssDNA 复合物解离的主要途径是首先与额外的 SSB 结合，然后解离。不同长度 ssDNA 的结合数据的比较没有给出四聚体之间协同性的证据。使用分析超速离心确定标记的SSB2.dT(70)在高离子强度(200mM NaCl)下的解离常数为1.1μM。测试了较短长度的单链DNA的结合：只有当长度减少到20个碱基时，亲和力才会显着降低。
A single-stranded DNA binding protein (SSB), labeled with a fluorophore, interacts With single-stranded DNA (ssDNA), giving a 6-fold increase in fluorescence. The labeled protein is the adduct of the G26C mutant of the homotetrameric SSB from Escherichia coli and a diethylaminocoumarin {N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide}. This adduct can be used to assay production of ssDNA during separation of double-stranded DNA by helicases. To use this probe effectively, as well as to investigate the interaction between ssDNA and SSB, the fluorescent SSB has been used to develop the kinetic mechanism by which the protein and ssDNA associate and dissociate. Under conditions where similar to 70 base lengths of ssDNA wrap around the tetramer, initial association is relatively simple and rapid, possibly diffusion-controlled. The kinetics are similar for a 70-base length of ssDNA, which binds one tetramer, and poly(dT), which could bind several. Under some conditions (high SSB and/or low ionic strength), a second tetramer binds to each 70-base length, but at a rate 2 orders of magnitude slower than the rate of binding of the first tetramer. Dissociation kinetics are complex and greatly accelerated by the presence of free wild-type SSB. The main route of dissociation of the fluorescent SSB.ssDNA complex is via association first with an additional SSB and then dissociation. Comparison of binding data with different lengths of ssDNA gave no evidence of cooperativity between tetramers. Analytical ultracentrifugation was used to determine the dissociation constant for labeled SSB2.dT(70) to be 1.1 mu M at a high ionic strength (200 mM NaCl). Shorter lengths of ssDNA were tested for binding: only when the length is reduced to 20 bases is the affinity significantly reduced.