Insertion sequence (IS)-excision enhancer (IEE) promotes the excision of ISs in the genome of enterohemorrhagic Escherichia coli O157. Because IEE-dependent IS excision occurs in the presence of transposase, the process of IS transposition may be involved in IS excision; however, little is understood about the molecular mechanisms of IS excision. Our in vitro analysis revealed that IEE exhibits DNA-dependent ATPase activity, which is activated by branched DNA. IEE also catalyzes the branch migration of fork-structured DNA. These results suggest that IEE remodels branched structures of the IS transposition intermediate. Sequence analysis of recombination sites in IS-excision products suggested that microhomologous sequences near the ends of the IS are involved in IS excision. IEE promoted microhomology-mediated end joining (MMEJ), in which base pairing between 6-nucleotides complementary ends of two 3′-protruding DNAs and subsequent elongation of the paired DNA strand occurred. IS-excision frequencies were significantly decreased in cells producing IEE mutants that had lost either branch migration or MMEJ activity, which suggests that these activities of IEE are required for IS excision. Based on our results, we propose a model for IS excision triggered by IEE and transposase.

中文翻译：

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催化分支迁移并促进微同源介导的末端连接的蛋白质增强了插入序列切除

插入序列（IS）-切除增强子（IEE）促进肠出血性大肠杆菌O157基因组中 IS 的切除。由于IEE依赖的IS切除发生在转座酶存在的情况下，因此IS转座过程可能参与IS切除；然而，人们对 IS 切除的分子机制知之甚少。我们的体外分析表明，IEE 表现出 DNA 依赖性 ATP 酶活性，该活性由分支 DNA 激活。 IEE 还催化叉状结构 DNA 的分支迁移。这些结果表明 IEE 重塑了 IS 转座中间体的分支结构。 IS 切除产物中重组位点的序列分析表明，IS 末端附近的微同源序列参与了 IS 切除。 IEE 促进了微同源介导的末端连接 (MMEJ)，其中两个 3' 突出 DNA 的 6 核苷酸互补末端之间发生碱基配对，并随后发生配对 DNA 链的延伸。在产生失去分支迁移或 MMEJ 活性的 IEE 突变体的细胞中，IS 切除频率显着降低，这表明 IEE 的这些活性是 IS 切除所必需的。根据我们的结果，我们提出了一个由 IEE 和转座酶触发的 IS 切除模型。