Bacterial DNA stability, Genome biology department, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France.
Evolution and maintenance of circular chromosomes, Genome biology department, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France.
PLoS Genet. 2018 Mar 9;14(3):e1007256. doi: 10.1371/journal.pgen.1007256. eCollection 2018 Mar.
It was recently reported that the recBC mutants of Escherichia coli, deficient for DNA double-strand break (DSB) repair, have a decreased copy number of their terminus region. We previously showed that this deficit resulted from DNA loss after post-replicative breakage of one of the two sister-chromosome termini at cell division. A viable cell and a dead cell devoid of terminus region were thus produced and, intriguingly, the reaction was transmitted to the following generations. Using genome marker frequency profiling and observation by microscopy of specific DNA loci within the terminus, we reveal here the origin of this phenomenon. We observed that terminus DNA loss was reduced in a recA mutant by the double-strand DNA degradation activity of RecBCD. The terminus-less cell produced at the first cell division was less prone to divide than the one produced at the next generation. DNA loss was not heritable if the chromosome was linearized in the terminus and occurred at chromosome termini that were unable to segregate after replication. We propose that in a recB mutant replication fork breakage results in the persistence of a linear DNA tail attached to a circular chromosome. Segregation of the linear and circular parts of this "σ-replicating chromosome" causes terminus DNA breakage during cell division. One daughter cell inherits a truncated linear chromosome and is not viable. The other inherits a circular chromosome attached to a linear tail ending in the chromosome terminus. Replication extends this tail, while degradation of its extremity results in terminus DNA loss. Repeated generation and segregation of new σ-replicating chromosomes explains the heritability of post-replicative breakage. Our results allow us to determine that in E. coli at each generation, 18% of cells are subject to replication fork breakage at dispersed, potentially random, chromosomal locations.
最近有报道称，缺乏 DNA 双链断裂 (DSB) 修复的大肠杆菌 recBC 突变体，其末端区域的拷贝数减少。我们之前曾表明，这种缺陷是由于细胞分裂时两条姐妹染色单体末端之一的复制后断裂导致的 DNA 丢失。因此，产生了一个有活力的细胞和一个没有末端区域的死细胞，而且，有趣的是，这种反应会传递到下一代。通过基因组标记频率分析和在特定 DNA 位观察末端，我们揭示了这种现象的起源。我们观察到，在 recA 突变体中，RecBCD 的双链 DNA 降解活性降低了末端 DNA 的丢失。在第一次细胞分裂中产生的无末端细胞比在下一次细胞分裂中产生的无末端细胞更不容易分裂。如果染色体在线粒体末端线性化，并且在复制后无法分离，那么染色体丢失就不会遗传。我们提出，在 recB 突变体中，复制叉断裂导致线性 DNA 尾巴附着在环状染色体上的持续存在。这种“σ复制染色体”的线性和环状部分的分离导致细胞分裂期间末端 DNA 断裂。一个子细胞继承了一个截断的线性染色体，因此无法存活。另一个子细胞继承了一个附着在线性尾部末端的环状染色体，该尾部末端位于染色体末端。复制延伸了这条尾巴，而其末端的降解导致末端 DNA 丢失。新的 σ 复制染色体的反复产生和分离解释了复制后断裂的遗传性。我们的结果使我们能够确定，在大肠杆菌中，每一代有 18%的细胞在分散的、潜在随机的染色体位置发生复制叉断裂。
