研究解析 T 修饰噬菌体 DNA 聚合酶偶联复制与尿嘧啶修复的结构基础
Structural basis for coupled DNA replication and uracil repair by a family of DNA polymerases from phages with T-modified genomes
研究者在携带 T 碱基修饰基因组的噬菌体中鉴定出一类新的 A 家族 DNA 聚合酶，这类酶与有活性的 UDG 结构域融合，并带有其他大片段插入。团队用冷冻电镜解析了来自 YerA41 和 phiLo 噬菌体的两个成员在多种功能与构象状态下的原子结构，显示这些大型聚合酶如何作为复制型 DNA 聚合酶工作，以及如何通过 UDG 结构域在复制叉前识别 dU 碱基、生成无碱基位点，并优先以掺入 dA 的方式越过。
来源：Nucleic Acids Research · doi.org
Replicative DNA polymerases (DNAPs) play a critical role in genome duplication, ensuring the accurate transmission of genetic information in all domains of life. DNAPs are also essential for DNA viruses, as well as bacteriophages, where a uracil-DNA glycosylase (UDG) is often encoded in trans or is present in the DNAP gene. Here, we identify a new group of A-family DNAPs in phages that are fused to an active UDG domain and contain other large insertions. Several members of this subfamily, including phages SP-15 and YerA41, are known to be hypermodified on their thymidine base through glycosylation of a linker attached to the C5 position of thymine. We determined the atomic cryo-EM structures for two of its members in various active functional and conformational states, from YerA41 and phiLo phages. The structures reveal how these large DNAPs function as replicative DNAPs, and how they can recognize dU bases ahead of the replication fork through their UDG domain, generating abasic sites that are preferentially bypassed through dA incorporation. Such dU errors would need to be eliminated to preserve phage genome integrity. Additionally, we report the co-occurrence of both an X-family DNAP and a DNA ligase in the corresponding phage genomes and show that both enzymes can execute the final steps of base excision repair most probably on-the-fly during replication, revealing a DNA-replication-coupled repair process. The structures of these large polymerases can serve as a model of other large platform DNAPs that can recruit additional enzymatic activities necessary for phage or virus survival.