Binding Specificity of Escherichia coli Single-Stranded DNA Binding Protein for the χ Subunit of DNA pol III Holoenzyme and PriA Helicase
Binding Specificity of Escherichia coli Single-Stranded DNA Binding Protein for the χ Subunit of DNA pol III Holoenzyme and PriA Helicase
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DOI：
10.1021/bi100069s
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发表时间：
2010-05-04
期刊：
影响因子：
2.9
通讯作者：
Lohman, Timothy M.
中科院分区：
文献类型：
--
作者：
Kozlov, Alexander G.;Jezewska, Maria J.;Lohman, Timothy M.
Escherichia Call单链DNA结合蛋白(SSB)通过其与单链DNA的高亲和力相互作用，以及通过其非结构的C末端与许多其他蛋白质的相互作用，在DNA代谢中发挥核心作用。虽然SSB与至少14种其他蛋白质相互作用，但人们还不知道SSB如何招募一种蛋白质而不是另一种蛋白质来发挥特定的代谢作用。为了探索这些相互作用的特异性，我们使用等温滴定量热法研究了SSB与两种对DNA复制至关重要的大肠杆菌蛋白-DNA聚合酶Ill全酶的x亚基和PrA解旋酶的结合热力学。我们发现，SSB四聚体可以主要通过与保守的SSB C末端尾巴(SSB-Ct)中最后9个类似氨基酸的相互作用，结合多达4个蛋白质分子。我们观察到一个分离的SSB-Ct多肽与PriA结合的内在特异性主要是由于一个更有利的焓成分。PriA和chi与SSB的亲和力也弱于与SSB-Ct的亲和力，表明SSB蛋白核心具有抑制作用。虽然SSB与单链DNA的结合亲和力增强，但对PriA的影响更大，表明SSB对PriA的特异性进一步增强。这些结果还表明，也与SSB相互作用的DNA结合蛋白，如PriA，可以利用这种相互作用，通过首先与SSB的C末端相互作用来获得与SSDNA的相互作用。
The Escherichia call single-stranded DNA binding protein (SSB) plays a central role in DNA metabolism through its high affinity interactions with ssDNA, as well as its interactions with numerous other proteins via its unstructured C-termini. Although SSB interacts with at least 14 other proteins, it is not understood how SSB might recruit one protein over another for a particular metabolic role. To probe the specificity of these interactions, we have used isothermal titration calorimetry to examine the thermodynamics of binding of SSB to two E. coli proteins important for DNA replication, the x subunit of DNA polymerase Ill holoenzyme and the PriA helicase. We find that an SSB tetramer can bind up to four molecules of either protein primarily via interactions with the last similar to 9 amino acids in the conserved SSB C-terminal tails (SSB-Ct). We observe intrinsic specificity for the binding of an isolated SSB-Ct peptide to PriA over chi due primarily to a more favorable enthalpic component. PriA and chi also bind with weaker affinity to SSB (in the absence of ssDNA) than to isolated SSB-Ct peptides, indicating an inhibitory effect of the SSB protein core. Although the binding affinity of SSB for both chi and PriA is enhanced if SSB is prebound to ssDNA, this effect is larger with PriA indicating a further enhancement of SSB specificity for PriA. These results also suggest that DNA binding proteins such as PriA, which also interact with SSB, could use this interaction to gain access to ssDNA by first interacting with the SSB C-termini.