Chromosomal DNA replication is a fundamental process of life, involving the assembly of complex machinery and dynamic regulation. In this study, we reconstructed a bacterial replication module (pRC) by artificially clustering 23 genes involved in DNA replication and sequentially deleting these genes from their naturally scattered loci on the chromosome of Escherichia coli. The integration of pRC into the chromosome, moving from positions farther away to close to the replication origin, leads to an enhanced efficiency in DNA synthesis, varying from lower to higher. Strains containing replication modules exhibited increased DNA replication by accelerating the replication fork movement and initiating chromosomal replication earlier in the replication cycle. The minimized module pRC16, containing only replisome and elongation encoding genes, exhibited chromosomal DNA replication efficiency comparable to that of pRC. The replication module demonstrated robust and rapid DNA replication, regardless of growth conditions. Moreover, the replication module is plug-and-play, and integrating it into Mb-sized extrachromosomal plasmids improves their genetic stability. Our findings indicate that DNA replication, being a fundamental life process, can be artificially reconstructed into replication functional modules. This suggests potential applications in DNA replication and the construction of synthetic modular genomes.

中文翻译：

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重建稳健的细菌复制模块


染色体 DNA 复制是生命的基本过程，涉及复杂机制的组装和动态调控。在这项研究中，我们通过人工聚集 23 个参与 DNA 复制的基因并依次从大肠杆菌染色体上自然分散的基因座中删除这些基因来重建细菌复制模块 （pRC）。pRC 整合到染色体中，从更远的位置移动到靠近复制起点的位置，导致 DNA 合成效率的提高，从低到高不等。含有复制模块的菌株通过加速复制叉运动并在复制周期的早期启动染色体复制来表现出 DNA 复制增加。最小化模块 pRC16 仅包含复制体和延伸编码基因，表现出与 pRC 相当的染色体 DNA 复制效率。无论生长条件如何，复制模块都表现出稳健和快速的 DNA 复制。此外，复制模块是即插即用的，将其整合到 Mb 大小的染色体外质粒中可以提高它们的遗传稳定性。我们的研究结果表明，DNA 复制作为一个基本的生命过程，可以人工重建为复制功能模块。这表明在 DNA 复制和合成模块化基因组构建方面的潜在应用。