Replication of the Escherichia coli chromosome in RNase HI-deficient cells: multiple initiation regions and fork dynamics
Replication of the Escherichia coli chromosome in RNase HI-deficient cells: multiple initiation regions and fork dynamics
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DOI：
10.1111/mmi.12440
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发表时间：
2014-01-01
影响因子：
3.6
通讯作者：
Kreuzer, Kenneth N.
中科院分区：
文献类型：
--
作者：
Maduike, Nkabuije Z.;Tehranchi, Ashley K.;Kreuzer, Kenneth N.
在大肠杆菌中的DNA复制通常是由单一起始点ORIC启动的，依赖于起始蛋白DNAA。然而，复制可以在染色体上的其他地方的多个异位Orik位点上启动。遗传证据表明，Orik的起始依赖于RNA-DNA杂交物(R-环)，这些R-环通常被RNase HI等酶去除，以防止Orik在正常生长过程中失火。在RNaseHI缺陷突变体中，存在从Orik位点的起始，在某些不寻常的条件下，也可能发生在野生型细胞中。尽管之前有过工作，但Orik的位置及其对基因组稳定性的影响仍不清楚。我们结合了2D凝胶电泳法和全基因组方法来绘制全基因组范围的Orik位置图。不同RNase HI缺陷株的DNA拷贝数图谱包含多个峰，通常位于一致的位置，以识别候选的Orik位点。RNase HI蛋白的去除也会导致复制分叉迁移模式的全球变化，通常与正常复制方向相反，并可能导致真核生物样的复制分叉合并。我们的结果对基因组稳定性有影响，为RNase HI缺陷如何导致R环介导的转录-复制冲突以及末端陷阱区域外的Ter位点和核糖体操纵子的不适当复制停滞或阻断提供了新的理解。
DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on RNA-DNA hybrids (R-loops), which are normally removed by enzymes such as RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The DNA copy number profiles of various RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for genome stability, offering a new understanding of how RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons.