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）中的科学家研究了将突变引入受损DNA的机制。现在众所周知，许多长期与诱变过程有关的蛋白质实际上是低保真DNA聚合酶，在称为Translesion DNA合成（TLS）的过程中可能遍历受损的DNA。 大肠杆菌中大多数损伤诱导的（SOS）诱变发生在DNA聚合酶V被RECA核蛋白丝（RECA*）激活时，催化TLS。在SOS反应过程中，RECA*在同源重组以及介导Lexa和Umud裂解中的生物学功能已得到充分了解。 相比之下，RECA*在依赖Pol V依赖性诱变TLS中的生化作用仍然很差。 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对于TLS。作为与南加州大学迈伦·古德曼（Myron Goodman）的合作研究的一部分，我们谈到了迄今为​​止，reca*在依赖POLV依赖性SOS诱变中的神秘作用。我们证明，reca*将单个Reca-ATP从其DNA 3'-End转移到Free Pol V（Umud'2C），形成一个活性型肌电组（pol vmut），并用组成Umud'c-reca-atp。在没有RECA*的情况下，pol Vmut催化TL，并在与DNA解离时迅速停用。在没有DNA合成的情况下，失活的发生较慢，同时将RECA-ATP保留在复合物中。 Pol Vmut的重新激活是由RECA*替换RECA-ATP触发的。因此，RECA*在SOS诱变中的主要作用是将RECA-ATP转移到Pol V中，以便生成活性的型突击型复合物以进行trans术合成。