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

基因组完整性实验室（LGI）的科学家研究了突变引入受损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，其可以在称为translesion DNA synthesis（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DNA聚合酶V，由RecA核蛋白丝（RecA *）激活，催化TLS时。RecA * 在同源重组中以及在SOS应答期间介导莱克萨和UmuD切割中的生物学功能是很好理解的。 相比之下，RecA * 在pol V依赖性致突变TLS中的生化作用仍然很难表征。关于RecA * 在TLS中的作用的建议已经从将UmuD 'C定位在邻近病变的引物/模板DNA上，发展到涉及通过前进的pol V置换模板上的RecA * 细丝的动态相互作用，发展到其中RecA * 不需要顺式位于被复制的模板链上，而是可以在单独的ssDNA链上组装以反式激活pol V用于TLS的模型。作为与Myron Goodman（南加州大学）合作研究的一部分，我们解决了迄今为止RecA * 在polV依赖性SOS诱变中的神秘作用。我们证明了RecA * 将单个RecA-ATP从其DNA 3 '端化学计量地转移到游离pol V（UmuD' 2C）以形成具有组成UmuD 'C-RecA-ATP的活性突变体（pol VMut）。Pol VMut在不存在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，同时在复合物中保留RecA-ATP。 通过从RecA * 替换RecA-ATP来触发pol VMut的再激活。因此，RecA * 在SOS突变中的主要作用是将RecA-ATP转移到pol V，从而产生活性突变体复合物用于跨损伤合成。

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.