Department of Chemistry, The Scripps Research Institute , La Jolla, California 92037, United States.
J Am Chem Soc. 2018 Jan 17;140(2):758-765. doi: 10.1021/jacs.7b11488. Epub 2018 Jan 8.
Semisynthetic organisms (SSOs) created from Escherichia coli can replicate a plasmid containing an unnatural base pair (UBP) formed between the synthetic nucleosides dNaM and dTPT3 (dNaM-dTPT3) when the corresponding unnatural triphosphates are imported via expression of a nucleoside triphosphate transporter. The UBP can also be transcribed and used to translate proteins containing unnatural amino acids. However, UBPs are not well retained in all sequences, limiting the information that can be encoded, and are invariably lost upon extended growth. Here we explore the contributions of the E. coli DNA replication and repair machinery to the propagation of DNA containing dNaM-dTPT3 and show that replication by DNA polymerase III, supplemented with the activity of polymerase II and methyl-directed mismatch repair contribute to retention of the UBP and that recombinational repair of stalled forks is responsible for the majority of its loss. This work elucidates fundamental aspects of how bacteria replicate DNA and we use this information to reprogram the replisome of the SSO for increased UBP retention, which then allowed for the first time the construction of SSOs harboring a UBP in their chromosome.
半合成生物体（SSO）可由大肠杆菌构建，当表达核苷酸三磷酸转运蛋白以导入相应的非天然三磷酸时，能够复制含有由合成核苷 dNaM 和 dTPT3（dNaM-dTPT3）之间形成的非天然碱基对（UBP）的质粒。该 UBP 还可以转录并用于翻译含有非天然氨基酸的蛋白质。然而，UBP 在所有序列中都不能很好地保留，限制了可编码的信息量，并且在长时间生长后总会丢失。在这里，我们研究了大肠杆菌 DNA 复制和修复机制对含有 dNaM-dTPT3 的 DNA 进行复制的贡献，并表明 DNA 聚合酶 III 的复制，辅以聚合酶 II 和甲基导向的错配修复的活性，有助于 UBP 的保留，而叉状stall 的重组修复是其丢失的主要原因。这项工作阐明了细菌复制 DNA 的基本方面，我们利用这些信息重新编程 SSO 的复制体以提高 UBP 的保留率，从而首次允许在其染色体中构建含有 UBP 的 SSO。
