DNA Replication, Repair, and Mutagenesis In Eukaryotic And Prokaryotic Cells

真核和原核细胞中的 DNA 复制、修复和诱变

• 批准号: 8149277
• 负责人: ROGER WOODGATE
• 金额: $ 247.63万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149277
• 关键词: Address; DNA; DNA Damage; DNA biosynthesis; DNA-Directed DNA Polymerase; Escherichia coli; Family; Filament; Human; Molecular; Mutagenesis; Pathologic Mutagenesis; Pathway interactions; Process; Prokaryotic Cells; Son of Sevenless Proteins; base; in vivo; repaired

Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into damaged DNA. It is now known that many of the proteins long implicated in the mutagenic process are, in fact, low-fidelity DNA polymerases that can traverse damaged DNA in a process termed translesion DNA synthesis (TLS). Most damage-induced (SOS) mutagenesis in Escherichia coli occurs when DNA polymerase V, activated by a RecA nucleoprotein filament (RecA*), catalyzes TLS. The biological functions of RecA* in homologous recombination and in mediating LexA and UmuD cleavage during the SOS response are well understood. In contrast, the biochemical role of RecA* in pol V-dependent mutagenic TLS remains poorly characterized. Proposals for the role of RecA* in TLS have evolved from positioning UmuD'C on primer/template DNA proximal to a lesion, to a dynamic interaction involving displacement of RecA* filaments on the template by an advancing pol V, to a model in which RecA* need not be located in cis on the template strand being copied, but can instead assemble on a separate ssDNA strand to transactivate pol V for TLS. As part of a collaborative study with Myron Goodman (University of Southern California), we addressed the hitherto enigmatic role of RecA* in polV-dependent SOS mutagenesis. We demonstrated that RecA* transfers a single RecA-ATP stoichiometrically from its DNA 3'-end to free pol V (UmuD'2C) to form an active mutasome (pol VMut) with the composition UmuD'C-RecA-ATP. Pol VMut catalyzes TLS in the absence of RecA* and deactivates rapidly upon dissociation from DNA. Deactivation occurs more slowly in the absence of DNA synthesis, while retaining RecA-ATP in the complex. Reactivation of pol VMut is triggered by replacement of RecA-ATP from RecA*. Thus, the principal role of RecA* in SOS mutagenesis is to transfer RecA-ATP to pol V, so as to generate active mutasomal complex for translesion synthesis.

基因组完整性实验室 (LGI) 的科学家研究将突变引入受损 DNA 的机制。现在已知，许多长期参与诱变过程的蛋白质实际上是低保真度 DNA 聚合酶，它们可以在称为跨损伤 DNA 合成 (TLS) 的过程中穿过受损的 DNA。 大肠杆菌中大多数损伤诱导 (SOS) 突变发生在 RecA 核蛋白丝 (RecA*) 激活的 DNA 聚合酶 V 催化 TLS 时。 RecA* 在同源重组以及在 SOS 响应期间介导 LexA 和 UmuD 裂解中的生物学功能已被充分了解。 相比之下，RecA* 在 pol V 依赖性诱变 TLS 中的生化作用仍然知之甚少。关于 RecA* 在 TLS 中的作用的建议已经从将 UmuD'C 定位在靠近病变的引物/模板 DNA 上，发展到涉及模板​​上 RecA* 丝通过前进的 pol V 进行位移的动态相互作用，再到 RecA* 不需要位于被复制的模板链上的顺式位置，而是可以组装在单独的 ssDNA 链上以反式激活 pol V 的模型 对于 TLS。作为与 Myron Goodman（南加州大学）合作研究的一部分，我们解决了 RecA* 在 polV 依赖性 SOS 诱变中迄今神秘的作用。我们证明 RecA* 将单个 RecA-ATP 按化学计量从其 DNA 3' 端转移到游离 pol V (UmuD'2C)，以形成具有 UmuD'C-RecA-ATP 组合物的活性变体 (pol VMut)。 Pol VMut 在没有 RecA* 的情况下催化 TLS，并在与 DNA 解离后迅速失活。在没有 DNA 合成的情况下，失活发生得更慢，同时将 RecA-ATP 保留在复合物中。 pol VMut 的重新激活是通过从 RecA* 替换 RecA-ATP 来触发的。因此，RecA*在SOS诱变中的主要作用是将RecA-ATP转移至pol V，从而产生用于跨损伤合成的活性变体复合物。