Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli.
Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli.
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DOI：
10.1093/g3journal/jkac295
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发表时间：
2022-12-01
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在大肠杆菌中，DNA复制复合体(复制体)与损伤的DNA或紧密结合在染色体上的蛋白质之间的碰撞导致复制体在每个细胞周期至少提前解离一次。如果不加以修复，这些事件会产生不完全复制的染色体，不能正确地分割成子细胞。因此，DNA复制重新启动，即在过早终止的位置重新加载复制体的过程，在大肠杆菌和其他细菌中是必不可少的。在大肠杆菌中已经确定了三种复制重新启动途径：PriA/Prib，PriA/PrIC和PrIC/Rep.复制重新启动与其他基因组维持途径之间的遗传相互作用已经定义了有限的数量，但将复制重新启动反应放在更广泛的细胞背景下的系统研究尚未进行。我们已经利用转座子插入测序来确定DNA复制重新启动途径和其他细胞系统之间的新的遗传相互作用。已知的与Prib复制重新启动基因(唯一参与PriA/Prib途径)的遗传相互作用已被证实，并发现了几种新的Prib相互作用。对Prib及其遗传伙伴进行的有针对性的遗传和基于成像的实验显示，在DAM、REP、rdgC、LexA或Pola突变的菌株中，双链DNA断裂显著积累。调节RecA重组酶的活性部分抑制了ΔPrib细胞中RdgC或LexA突变的有害影响。综上所述，我们的结果强调了几个基因在双链DNA破坏动态平衡中的作用，并定义了一个遗传网络，该网络促进了在大肠杆菌中PrA/Prib介导的DNA复制重新启动的上游DNA修复/处理。
Collisions between DNA replication complexes (replisomes) and impediments such as damaged DNA or proteins tightly bound to the chromosome lead to premature dissociation of replisomes at least once per cell cycle in Escherichia coli. Left unrepaired, these events produce incompletely replicated chromosomes that cannot be properly partitioned into daughter cells. DNA replication restart, the process that reloads replisomes at prematurely terminated sites, is therefore essential in E. coli and other bacteria. Three replication restart pathways have been identified in E. coli: PriA/PriB, PriA/PriC, and PriC/Rep. A limited number of genetic interactions between replication restart and other genome maintenance pathways have been defined, but a systematic study placing replication restart reactions in a broader cellular context has not been performed. We have utilized transposon-insertion sequencing to identify new genetic interactions between DNA replication restart pathways and other cellular systems. Known genetic interactors with the priB replication restart gene (uniquely involved in the PriA/PriB pathway) were confirmed and several novel priB interactions were discovered. Targeted genetic and imaging-based experiments with priB and its genetic partners revealed significant double-strand DNA break accumulation in strains with mutations in dam, rep, rdgC, lexA, or polA. Modulating the activity of the RecA recombinase partially suppressed the detrimental effects of rdgC or lexA mutations in ΔpriB cells. Taken together, our results highlight roles for several genes in double-strand DNA break homeostasis and define a genetic network that facilitates DNA repair/processing upstream of PriA/PriB-mediated DNA replication restart in E. coli.
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