RecG Directs DNA Synthesis during Double-Strand Break Repair.
RecG Directs DNA Synthesis during Double-Strand Break Repair.
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DOI：
10.1371/journal.pgen.1005799
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发表时间：
2016-02
期刊：
影响因子：
4.5
通讯作者：
Leach DR
中科院分区：
文献类型：
--
作者：
Azeroglu B;Mawer JS;Cockram CA;White MA;Hasan AM;Filatenkova M;Leach DR
同源重组提供了DNA双链断裂修复（DSBR）的机制，其需要完整的同源模板用于DNA合成。当与DSBR相关的DNA合成收敛时，断裂的DNA链被替换，修复是准确的。然而，如果建立了趋异DNA合成，则可能发生侧翼DNA的过度复制，并产生有害后果。大肠杆菌的RecG蛋白是一种解旋酶和移位酶，可以重新建模3-way和4-way DNA结构，如复制叉和霍利迪连接。然而，RecG在活细胞中的主要作用仍然难以捉摸。在这里，我们表明，在没有RecG的情况下，试图DSBR是伴随着在诱导的染色体DNA双链断裂的网站上的DNA复制的分歧。此外，在recG突变体中，在已知阻断复制叉的位点处产生DNA双链末端。这些双链末端也触发DSBR和该突变体的趋异DNA复制特征，这可以解释染色体末端区域的过度复制。先前在不存在RuvAB和RecG的情况下观察到的与解旋关节分子相关的DNA损失被解旋酶缺陷型PriA突变（priA 300）抑制，认为RecG的作用确保PriA正确结合在D环上以指导DNA复制，而不是解旋关节分子。这使得我们提出了一个修正的同源重组模型，其中RecG对分支中间体的重新建模在指导DNA合成中起着重要作用，从而保持基因组的稳定性。DNA双链断裂可通过同源重组精确修复。这种准确性是通过复制DNA的第二个完整拷贝上存在的正确遗传信息来确保的，通常是在DNA复制过程中产生的姐妹染色体。这意味着在重组过程中发生的DNA合成必须被引导以替换丢失或损坏的碱基对，而不是过度复制未损坏的染色体区域。在这里，我们调查的RecG缺乏DNA修复过程中的基因组后果后，位点特异性双链断裂引入两个同源E。coli染色体。我们的观察表明，RecG可以重新建模分支的重组中间体，以指导正确的结合PriA。这建立了会聚的复制叉，其取代DSBR位点处丢失的DNA，并防止侧翼DNA区域的过度复制。这使我们重新评估我们对大肠杆菌中同源重组途径的理解。大肠杆菌，并提出了一个模型，其中RecG在重组和DNA复制的界面处重塑分支中间体中起着至关重要的作用。
Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability. DNA double-strand breaks are accurately repaired by homologous recombination. This accuracy is ensured by copying the correct genetic information present on a second unbroken copy of the DNA, normally a sister chromosome that is generated during DNA replication. This implies that DNA synthesis occurring during recombination must be directed to replace lost or damaged base pairs but not to over-replicate undamaged chromosomal regions. Here, we investigate the genomic consequences of the absence of RecG during DNA repair following a site-specific double-strand break introduced in only one of two homologous E. coli chromosomes. Our observations suggest that RecG can re-model branched intermediates of recombination to direct the correct binding of PriA. This establishes converging replication forks that replace lost DNA at the site of DSBR and prevents over-replication of flanking DNA regions. This has led us to re-evaluate our understanding of the pathway of homologous recombination in E. coli and to propose a model in which RecG plays a critical role in remodelling branched intermediates at the interface of recombination and DNA replication.