Amundsen Susan K, Sharp Jake W, Smith Gerald R
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109.
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109
Genetics. 2016 Sep;204(1):139-52. doi: 10.1534/genetics.116.191056. Epub 2016 Jul 8.
RecBCD enzyme is a complex, three-subunit protein machine essential for the major pathway of DNA double-strand break repair and homologous recombination in Escherichia coli Upon encountering a Chi recombination-hotspot during DNA unwinding, RecBCD nicks DNA to produce a single-stranded DNA end onto which it loads RecA protein. Conformational changes that regulate RecBCD's helicase and nuclease activities are induced upon its interaction with Chi, defined historically as 5' GCTGGTGG 3'. Chi is thought to be recognized as single-stranded DNA passing through a tunnel in RecC. To define the Chi recognition-domain in RecC and thus the mechanism of the RecBCD-Chi interaction, we altered by random mutagenesis eight RecC amino acids lining the tunnel. We screened for loss of Chi activity with Chi at one site in bacteriophage λ. The 25 recC mutants analyzed thoroughly had undetectable or strongly reduced Chi-hotspot activity with previously reported Chi sites. Remarkably, most of these mutants had readily detectable, and some nearly wild-type, activity with Chi at newly generated Chi sites. Like wild-type RecBCD, these mutants had Chi activity that responded dramatically (up to fivefold, equivalent to Chi's hotspot activity) to nucleotide changes flanking 5' GCTGGTGG 3'. Thus, these and previously published RecC mutants thought to be Chi-recognition mutants are actually Chi context-dependence mutants. Our results fundamentally alter the view that Chi is a simple 8-bp sequence recognized by the RecC tunnel. We propose that Chi hotspots have dual nucleotide sequence interactions, with both the RecC tunnel and the RecB nuclease domain.
RecBCD酶是一种复杂的三聚体蛋白质机器，对大肠杆菌中DNA双链断裂修复和同源重组的主要途径至关重要。在DNA解旋过程中遇到Chi重组热点时，RecBCD会切割DNA以产生单链DNA末端，并在其上加载RecA蛋白。与Chi相互作用时会诱导调节RecBCD解旋酶和核酸酶活性的构象变化，Chi在历史上被定义为5' GCTGGTGG 3'。人们认为Chi被识别为通过RecC中一个通道的单链DNA。为了确定RecC中的Chi识别结构域以及RecBCD与Chi相互作用的机制，我们通过随机诱变改变了通道内衬的8个RecC氨基酸。我们在噬菌体λ的一个位点筛选了Chi活性丧失的情况。对25个recC突变体进行的深入分析表明，对于先前报道的Chi位点，它们的Chi热点活性无法检测到或大幅降低。值得注意的是，这些突变体中的大多数对于新产生的Chi位点的Chi具有易于检测到的活性，有些几乎具有野生型活性。与野生型RecBCD一样，这些突变体的Chi活性对5' GCTGGTGG 3'侧翼的核苷酸变化有显著响应（高达五倍，相当于Chi的热点活性）。因此，这些以及先前发表的被认为是Chi识别突变体的RecC突变体实际上是Chi上下文依赖性突变体。我们的结果从根本上改变了认为Chi是RecC通道识别的简单8碱基对序列的观点。我们提出，Chi热点具有双重核苷酸序列相互作用，与RecC通道和RecB核酸酶结构域都有关。
