Reed Tristan T, Kendal Abigail H, Wozniak Katherine J, Simmons Lyle A
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.
J Bacteriol. 2025 Jul 21:e0017525. doi: 10.1128/jb.00175-25.
DNA replication is regulated by factors that promote or inhibit initiation. In YabA is a negative regulator of replication initiation, while the newly identified kinase CcrZ is a positive regulator. The consequences of under-initiation or over-initiation of replication on genome stability remain unclear. In this work, we measure the origin-to-terminus ratio as a proxy for replication initiation activity. We show that Δ and several alleles under-initiate DNA replication, while ablation of or overproduction of CcrZ leads to over-initiation. We find that cells under-initiating DNA replication have few incidents of replication fork stress as determined by the low frequency formation of RecA-GFP foci compared with wild type. In contrast, cells over-initiating replication show levels of RecA-GFP foci formation analogous to cells directly challenged with DNA-damaging agents. We show that cells under-initiating and over-initiating DNA replication are both sensitive to mitomycin C, demonstrating that changes in replication initiation frequency cause an increase in sensitivity to genotoxic stress. With these results, we propose that cells under-initiating DNA replication are sensitive to DNA damage due to asynchronous DNA replication, leading to inefficient homologous recombination. In cells over-initiating replication, we propose that an increase in the number of replication forks leads to replication fork stress, which is further exacerbated by chromosomal DNA damage. Together, our study shows that DNA replication initiation frequency must be tightly controlled because changes in initiation influence replication fork fate and the capacity of cells to efficiently repair damage to their genetic material.IMPORTANCEThe regulation of DNA replication is fundamental to cell growth and cell cycle control. In eukaryotes, under-initiation or over-initiation leads to genome instability. In bacteria, it is unclear how changes in replication initiation frequency impact DNA replication status and genome integrity. We show that tight regulation of DNA replication initiation is critical for maintaining genome integrity. Cells over-initiating or under-initiating DNA replication are sensitive to DNA damage. Furthermore, cells over-initiating DNA replication experience replication fork stress at levels that phenocopy those observed in cells challenged with DNA damage from mitomycin C. Our results establish the critical importance of properly regulating DNA replication initiation frequency because an imbalance in initiation results in replication fork perturbations, deficiencies in DNA repair, and genome instability.
DNA复制受促进或抑制起始的因子调控。在[具体细菌名称]中，YabA是复制起始的负调控因子，而新鉴定的激酶CcrZ是正调控因子。复制起始不足或过度起始对基因组稳定性的影响仍不清楚。在这项研究中，我们测量起始点与终点的比例作为复制起始活性的指标。我们发现缺失[具体基因名称]和几个[具体等位基因名称]会导致DNA复制起始不足，而缺失[具体基因名称]或过量表达CcrZ会导致起始过度。我们发现，与野生型相比，DNA复制起始不足的细胞中RecA-GFP焦点形成频率较低，表明复制叉应激事件较少。相反，复制起始过度的细胞中RecA-GFP焦点形成水平与直接受到DNA损伤剂攻击的细胞相似。我们表明，DNA复制起始不足和过度的细胞对丝裂霉素C均敏感，这表明复制起始频率的变化会导致对基因毒性应激的敏感性增加。基于这些结果，我们提出，DNA复制起始不足的细胞由于DNA复制异步而对DNA损伤敏感，导致同源重组效率低下。在复制起始过度的细胞中，我们提出复制叉数量的增加会导致复制叉应激，而染色体DNA损伤会进一步加剧这种应激。总之，我们的研究表明，DNA复制起始频率必须受到严格控制，因为起始的变化会影响复制叉的命运以及细胞有效修复其遗传物质损伤的能力。
重要性
DNA复制的调控对于细胞生长和细胞周期控制至关重要。在真核生物中，起始不足或过度会导致基因组不稳定。在细菌中，尚不清楚复制起始频率的变化如何影响DNA复制状态和基因组完整性。我们表明，严格调控DNA复制起始对于维持基因组完整性至关重要。复制起始过度或不足的细胞对DNA损伤敏感。此外，复制起始过度的细胞经历的复制叉应激水平与受到丝裂霉素C DNA损伤攻击的细胞相似。我们的结果确立了正确调控DNA复制起始频率的至关重要性，因为起始失衡会导致复制叉扰动、DNA修复缺陷和基因组不稳定。
