Type III restriction is alleviated by bacteriophage (RecE) homologous recombination function but enhanced by bacterial (RecBCD) function
Type III restriction is alleviated by bacteriophage (RecE) homologous recombination function but enhanced by bacterial (RecBCD) function
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DOI：
10.1128/jb.187.21.7362-7373.2005
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发表时间：
2005-11-01
影响因子：
3.2
通讯作者：
Kobayashi, I
中科院分区：
文献类型：
--
作者：
Handa, N;Kobayashi, I
以前的工作已经证明，限制酶产生的DNA断裂通过Lambdoid噬菌体lambda和Rae的作用与完整的、同源的DNA区域进行同源重组并被修复。在目前的工作中，我们研究了在细菌细胞中表达的噬菌体功能对在简单的空斑形成试验中限制感染测试噬菌体的影响。携带EcoRI的大肠杆菌宿主上的空斑形成效率不会因RAE原噬菌体的存在而增加--这可能是因为，在空斑分析的单一感染条件下，断裂的噬菌体DNA无法找到可与之重组的同源物。然而，令我们惊讶的是，我们发现，在存在III型限制系统EcoP1或EcoP15的情况下，噬菌体介导的RAE原噬菌体的同源重组功能Ress和RECT提高了斑块形成的效率。与类型I限制缓解不同，该类型III限制缓解不依赖于更大的RAE。另一方面，细菌RecBCD-同源重组功能增强了III型限制。这些结果导致我们假设，III型限制性内切酶的作用发生在体内复制或复制DNA上，并在非等位基因位点留下断裂的子代DNA，噬菌体介导的同源重组从它们中重组出完整的DNA，而RecBCD外切酶通过降解限制性断裂来阻止这种修复。
Previous works have demonstrated that DNA breaks generated by restriction enzymes stimulate, and are repaired by, homologous recombination with an intact, homologous DNA region through the function of lambdoid bacteriophages lambda and Rae. In the present work, we examined the effect of bacteriophage functions, expressed in bacterial cells, on restriction of an infecting tester phage in a simple plaque formation assay. The efficiency of plaque formation on an Escherichia coli host carrying EcoRI, a type II restriction system, is not increased by the presence of Rae prophage-presumably because, under the single-infection conditions of the plaque assay, a broken phage DNA cannot find a homologue with which to recombine. To our surprise, however, we found that the efficiency of plaque formation in the presence of a type III restriction system, EcoP1 or EcoP15, is increased by the bacteriophage-mediated homologous recombination functions recE and recT of Rae prophage. This type III restriction alleviation does not depend on lar on Rae, unlike type I restriction alleviation. On the other hand, bacterial RecBCD-homologous recombination function enhances type III restriction. These results led us to hypothesize that the action of type III restriction enzymes takes place on replicated or replicating DNA in vivo and leaves daughter DNAs with breaks at nonallelic sites, that bacteriophage-mediated homologous recombination reconstitutes an intact DNA from them, and that RecBCD exonuclease blocks this repair by degradation from the restriction breaks.