Crystal structure of the Red C-terminal domain in complex with Exonuclease reveals an unexpected homology with Orf and an interaction with Escherichia coli single stranded DNA binding protein
Crystal structure of the Red C-terminal domain in complex with Exonuclease reveals an unexpected homology with Orf and an interaction with Escherichia coli single stranded DNA binding protein
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DOI：
10.1093/nar/gky1309
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发表时间：
2019-02-28
影响因子：
14.9
通讯作者：
Bell, Charles E.
中科院分区：
文献类型：
--
作者：
Caldwell, Brian J.;Zakharova, Ekaterina;Bell, Charles E.
噬菌体编码DNA重组系统，其包括5-3核酸外切酶（Exo）和单链退火蛋白（Red）。这两种蛋白质形成复合物，被认为介导Red直接加载到由Exo产生的单链3-突出端上。在这里，我们提出了一个2.3埃的晶体结构的外三聚体绑定到三个副本的红色C-末端结构域（CTD）。在界面的疏水核心处的残基的突变破坏体外复合物的形成并损害体内重组。RedCTD形成一个三螺旋束，与噬菌体Orf具有意想不到的结构同源性，Orf是一种与E. coli单链DNA结合蛋白（SSB）作为重组介体。基于这种关系，我们发现，红色结合全长SSB，并对应于其9个C-末端残基的肽，在相互作用，需要CTD。这些结果表明CTD的双重作用，首先是结合Exo以促进Red直接加载到初始单链DNA（ssDNA）的3-突出端，其次是结合SSB以促进突出端退火到复制叉处的SSB包被的ssDNA。
Bacteriophage encodes a DNA recombination system that includes a 5-3 exonuclease ( Exo) and a single strand annealing protein (Red). The two proteins form a complex that is thought to mediate loading of Red directly onto the single-stranded 3-overhang generated by Exo. Here, we present a 2.3 angstrom crystal structure of the Exo trimer bound to three copies of the Red C-terminal domain (CTD). Mutation of residues at the hydrophobic core of the interface disrupts complex formation in vitro and impairs recombination in vivo. The RedCTD forms a three-helix bundle with unexpected structural homology to phage Orf, a protein that binds to E. coli single-stranded DNA binding protein (SSB) to function as a recombination mediator. Based on this relationship, we found that Red binds to full-length SSB, and to a peptide corresponding to its nine C-terminal residues, in an interaction that requires the CTD. These results suggest a dual role of the CTD, first in binding to Exo to facilitate loading of Reddirectly onto the initial single-stranded DNA (ssDNA) at a 3-overhang, and second in binding to SSB to facilitate annealing of the overhang to SSB-coated ssDNA at the replication fork.