Chromosome demise in the wake of ligase-deficient replication
Chromosome demise in the wake of ligase-deficient replication
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DOI：
10.1111/j.1365-2958.2012.08076.x
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发表时间：
2012-06-01
影响因子：
3.6
通讯作者：
Kuzminov, Andrei
中科院分区：
文献类型：
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作者：
Kouzminova, Elena A.;Kuzminov, Andrei
细菌DNA连接酶是一种依赖于NAD+的酶，不同于真核生物的依赖于ATP的连接酶，是广谱抗菌药物的潜在靶点。然而，连接酶缺陷的DNA复制在此期间积累冈崎片段的染色体后果仍不清楚。以大肠杆菌最强的连接酶突变体igA251(Ts)为材料，从遗传和物理两个方面研究了连接酶缺陷的DNA复制。在这里，我们展示了在没有连接酶的情况下，复制在短暂的抵抗期后被杀死。我们发现，通过RecA、RecBCD、RuvABC和RecG进行的双链断裂修复解释了瞬时抗性，而不可修复的染色体断裂解释了随后的细胞死亡。值得注意的是，死亡主要是通过消除ExoV的线性DNA降解活性来防止的，这表明复制叉子后面的非等位双链断裂导致DNA降解，从而扩大到等位基因双链间隙。同步复制的标记频率分析显示，连接酶缺陷的叉子停滞，随后没有连接酶合成的DNA降解。复制叉子后面未密封的缺口首先转化为可修复的双链断裂，然后转化为不可修复的双链缺口，这种机制可能是任何DNA损伤治疗致命的背后原因。
Bacterial DNA ligases, NAD+-dependent enzymes, are distinct from eukaryotic ATP-dependent ligases, representing promising targets for broad-spectrum antimicrobials. Yet, the chromosomal consequences of ligase-deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase-deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double-strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non-allelic double-strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double-strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase-deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double-strand breaks and then into irreparable double-strand gaps may be behind lethality of any DNA damaging treatment.