一项用于测定来自大肠杆菌的单链DNA结合蛋白相对核酸结合亲和力的电子顺磁共振研究。

An EPR study to determine the relative nucleic acid binding affinity of single-stranded DNA-binding protein from Escherichia coli.

作者信息

Bobst E V, Perrino F W, Meyer R R, Bobst A M

机构信息

Department of Chemistry, University of Cincinnati, OH 45221.

出版信息

Biochim Biophys Acta. 1991 Jun 24;1078(2):199-207. doi: 10.1016/0167-4838(91)99010-p.

DOI:10.1016/0167-4838(91)99010-p

PMID:1648407

Abstract

A direct quantitative determination by EPR of the nucleic acid binding affinity relationship of the single-stranded DNA-binding protein (SSB) from Escherichia coli at close to physiological NaCl concentration is reported. Titrations of (DUAP, dT)n, an enzymatically spin-labeled (dT)n, with SSB in 20 mM Tris-HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton with either low (5 mM), intermediate (125 mM) or high 200 mM) NaCl content, reveal the formation of a high nucleic acid density complex with a binding stoichiometry (s) of 60 to 75 nucleotides per SSB tetramer. Reverse titrations, achieved by adding (DUAP, dT)n to SSB-containing solutions, form a low nucleic acid density complex with an s = 25 to 35 in the buffer with low NaCl content (5 mM NaCl). The complex with an s = 25 to 35 is converted to the high nucleic acid density complex by increasing the NaCl content to 200 mM. It is, therefore, metastable and forms only under reverse titration conditions in low NaCl. The relative apparent affinity constant Kapp of SSB for various unlabeled single-stranded nucleic acids was determined by EPR competition experiments with spin-labeled nucleic acids as macromolecular probes in the presence of the high nucleic acid density complex. The Kapp of SSB exhibits the greatest affinity for (dT)n as was previously found for T4 gene 32 protein (Bobst, A.M., Langemeier, P.W., Warwick-Koochaki, P.E., Bobst, E.V. and Ireland, J.C. (1982) J. Biol. Chem. 257, 6184) and gene 5 protein (Bobst, A.M., Ireland, J.C. and Bobst, E.V. (1984) J. Biol. Chem. 259, 2130) by EPR competition assays. In contrast, however, SSB does not display several orders of magnitude greater affinity for (dT)n than for other single stranded DNAs as is the case with both gene 5 and T4 gene 32 protein. The relative Kapp values for SSB in the above buffer with 125 mM NaCl are: Kapp(dT)n = 4KappfdDNA = 40Kapp(dA)n = 200Kapp(A)n.

摘要

本文报道了在接近生理NaCl浓度下，通过电子顺磁共振（EPR）对大肠杆菌单链DNA结合蛋白（SSB）的核酸结合亲和力关系进行的直接定量测定。在含有20 mM Tris-HCl（pH 8.1）、1 mM乙二胺四乙酸钠、0.1 mM二硫苏糖醇、10%（w/v）甘油、0.05% Triton且NaCl含量分别为低（5 mM）、中（125 mM）或高（200 mM）的缓冲液中，用SSB对酶促自旋标记的（dT）n（DUAP，dT）n进行滴定，结果显示形成了一种高核酸密度复合物，每个SSB四聚体的结合化学计量比（s）为60至75个核苷酸。通过向含SSB的溶液中添加（DUAP，dT）n进行反向滴定，在低NaCl含量（5 mM NaCl）的缓冲液中形成了一种低核酸密度复合物，其s = 25至35。通过将NaCl含量增加到200 mM，s = 25至35的复合物可转化为高核酸密度复合物。因此，它是亚稳态的，仅在低NaCl的反向滴定条件下形成。在高核酸密度复合物存在的情况下，以自旋标记的核酸作为大分子探针，通过EPR竞争实验测定了SSB对各种未标记单链核酸的相对表观亲和常数Kapp。正如之前通过EPR竞争分析发现T4基因32蛋白（Bobst，A.M.，Langemeier，P.W.，Warwick-Koochaki，P.E.，Bobst，E.V.和Ireland，J.C.（1982）J. Biol. Chem. 257，6184）和基因5蛋白（Bobst，A.M.，Ireland，J.C.和Bobst，E.V.（1984）J. Biol. Chem. 259，2130）那样，SSB对（dT）n表现出最大的亲和力。然而，与基因5和T4基因32蛋白不同的是，SSB对（dT）n的亲和力并不比其他单链DNA高几个数量级。在上述含有125 mM NaCl的缓冲液中，SSB的相对Kapp值为：Kapp（dT）n = 4Kapp fdDNA = 40Kapp（dA）n = 200Kapp（A）n。