Structural basis of Escherichia coli single-stranded DNA-binding protein stimulation of exonuclease I
Structural basis of Escherichia coli single-stranded DNA-binding protein stimulation of exonuclease I
复制标题
DOI：
10.1073/pnas.0800741105
复制
发表时间：
2008-07-08
影响因子：
11.1
通讯作者：
Keck, James L.
中科院分区：
文献类型：
--
作者：
Lu, Duo;Keck, James L.
细菌单链DNA结合蛋白（SSBs）在基因组生物学中起着重要的保护作用，可以保护ssDNA免受损伤，防止假DNA退火。ssDNA/SSB复合物远非惰性的，而是动态的DNA加工中心，许多不同的酶通过利用与SSB的直接相互作用获得基因组底物。在迄今为止检查的所有情况下，SSB的C末端（SSB-Ct）形成异源蛋白的对接位点。我们描述了包含SSB-Ct元件的肽和来自大肠杆菌的核酸外切酶I（ExoI）之间形成的复合物的2.7埃分辨率晶体结构。两个SSB-Ct肽与ExoI上的相邻位点结合。诱变研究表明，这些位点之一是重要的协会与SSB-Ct肽在溶液中和SSB刺激ExoI活性，而第二个没有可辨别的功能。这些研究确定了ExoI/SSB-Ct复合物的稳定性与ExoI活性的SSB刺激之间的相关性。此外，SSB的C末端内的突变产生不能刺激ExoI活性的变体，而SSB-Ct肽单独没有作用。总之，我们的研究结果表明，SSB通过招募酶到其底物来刺激ExoI，并为理解SSB在基因组维护中的组织作用提供了结构范例。
Bacterial single-stranded DNA (ssDNA)-binding proteins (SSBs) play essential protective roles in genome biology by shielding ssDNA from damage and preventing spurious DNA annealing. Far from being inert, ssDNA/SSB complexes are dynamic DNA processing centers where-many different enzymes gain access to genomic substrates by exploiting direct interactions with SSB. In all cases examined to date, the C terminus of SSB (SSB-Ct) forms the docking site for heterologous proteins. We describe the 2.7-angstrom-resolution crystal structure of a complex formed between a peptide comprising the SSB-Ct element and exonuclease I (ExoI) from Escherichia coli. Two SSB-Ct peptides bind to adjacent sites on ExoI. Mutagenesis studies indicate that one of these sites is important for association with the SSB-Ct peptide in solution and for SSB stimulation of ExoI activity, whereas the second has no discernable function. These studies identify a correlation between the stability of the ExoI/SSB-Ct complex and SSB-stimulation of ExoI activity. Furthermore, mutations within SSB's C terminus produce variants that fail to stimulate ExoI activity, whereas the SSB-Ct peptide alone has no effect. Together, our findings indicate that SSB stimulates ExoI by recruiting the enzyme to its substrate and provide a structural paradigm for understanding SSB's organizational role in genome maintenance.