ESCHERICHIA-COLI SINGLE-STRAND BINDING-PROTEIN FORMS MULTIPLE, DISTINCT COMPLEXES WITH SINGLE-STRANDED-DNA
ESCHERICHIA-COLI SINGLE-STRAND BINDING-PROTEIN FORMS MULTIPLE, DISTINCT COMPLEXES WITH SINGLE-STRANDED-DNA
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DOI：
10.1021/bi00372a003
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发表时间：
1986-12-02
期刊：
影响因子：
2.9
通讯作者：
LOHMAN, TM
中科院分区：
文献类型：
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作者：
BUJALOWSKI, W;LOHMAN, TM
大肠杆菌单链结合（SSB）蛋白与单链（ss）DNA相互作用的四种不同的结合模式已经基于定量滴定法被鉴定，所述定量滴定法监测在25和37 ℃下MgCl2和NaCl浓度范围内SSB蛋白与同聚多核苷酸聚（dT）结合时荧光的淬灭。C.这是第一次观察到单一蛋白质与DNA结合的多种结合模式。这些结果扩展了以前在NaCl（25 ℃）中进行的研究。C，pH8.1），其中观察到两种不同的SSB-ss DNA结合模式，每个结合的SSB四聚体具有33和65个核苷酸的位点大小[Lohman，T. M.，和Overman，L. B。（1985）J.Biol.Chem.260，3594 - 3603]。这些结合模式中的每一种在与ss DNA相互作用时封闭的核苷酸的数量上不同（即，站点大小）。沿着先前观察到的具有35 ±. 2和65。+-。每个四聚体3个核苷酸，第三种不同的结合模式，在25 ° C下。C，已被确定，拥有的网站大小为56。每个结合的SSB四聚体3个核苷酸，其在广泛的MgCl 2浓度范围内稳定。在37度。C时，观察到第四种结合模式，具有40 ± 1的位点大小。每个四聚体2个核苷酸，尽管这种模式仅在小范围的盐浓度上可观察到。每种结合模式的相对群体主要由溶液中低分子量阳离子（在这些实验中为Mg 2+和Na+）的电荷和浓度调制，表明在形成每种较高位点大小的SSB-DNA复合物时发生阳离子的净结合。Mg2+比Na+更有效地促进向更高位点尺寸结合模式的转变。在25度。C（pH 8.1），两种结合模式转变的中点在MgCl2中为0.6和64 mM，在NaCl中为17 mM和0.16 M;因此，所有三种SSB结合模式都可能在体内形成，因为这些盐浓度在估计发生在大肠杆菌中的范围内。杆菌这些转变也发生在用于体外复制和重组研究的相同MgCl2浓度范围内;因此，它们对于任何此类体外研究都具有明确的重要性。自从E. coli SSB蛋白是复制、重组和修复过程所必需的，因此，在此鉴定的不同结合模型有可能选择性地用于体内这些过程中的每一个。
Four distinct binding modes for the interaction of Escherichia coli single-strand binding (SSB) protein with single-stranded (ss) DNA have been identified on the basis of quantitative titrations that monitor the quenching of the SSB protein fluorescence upon binding to the homopolynucleotide poly(dT) over a range of MgCl2 and NaCl concentrations at 25 and 37.degree. C. This is the first observation of multiple binding modes for a single protein binding to DNA. These results extend previous studies performed in NaCl (25.degree. C, pH 8.1), in which two distinct SSB-ss DNA binding modes possessing site sizes of 33 and 65 nucleotides per bound SSB tetramer were observed [Lohman, T. M., and Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603]. Each of these binding modes differs in the number of nucleotides occluded upon interaction with ss DNA (i.e., site size). Along with the previously observed modes with site sizes of 35 .+-. 2 and 65 .+-. 3 nucleotides per tetramer, a third distinct binding mode, at 25.degree. C, has been identified, possessing a site size of 56 .+-. 3 nucleotides per bound SSB tetramer, which is stable over a wide range of MgCl2 concentrations. At 37.degree. C, a fourth binding mode is observed, possessing a site size of 40 .+-. 2 nucleotides per tetramer, although this mode is observable only over a small range of salt concentration. The relative populations of each binding mode are modulated primarily by the charge and concentration of low molecular weight cations in solution(Mg2+ and Na+ in these experiments), indicating tht a net binding of cations occurs upon formation of each of the higher site size SSB-DNA complexes. Mg2+ is much more effective than Na+ in facilitating the transitions to the higher site size binding modes. At 25.degree. C (pH 8.1) the two binding mode transitions have midpoints of 0.6 and 64 mM in MgCl2 and 17 mM and 0.16 M in NaCl; hence, all three SSB binding modes may form in vivo since these salt concentrations are within the range estimated to occur in E. coli. These transitions also occur within the same range of MgCl2 concentrations used for replication and recombination studies in vitro; hence, they are of definite importance for any such studies in vitro. Since the E. coli SSB protein is essential for replication, recombination, and repair process, it is possible that the different binding models identified here are used selectively in each of these processes in vivo.