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: 7968592
• 负责人: ROGER WOODGATE
• 金额: $ 278.72万
• 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/7968592
• 关键词: Address; Affect; Bacteria; Binding Sites; Biochemical; Biological Assay; Biological Process; California; Cells; Complex; DNA; DNA Damage; DNA Primers; DNA biosynthesis; DNA lesion; DNA polymerase V; DNA polymerase iota; DNA-Directed DNA Polymerase; Data; Dissociation; Enzymatic Biochemistry; Escherichia coli; Eukaryota; Exhibits; Exodeoxyribonuclease I; Family; Filament; Fluorescence; Genome; Genomics; Goals; Human; In Vitro; Investigation; Label; Laboratories; Lesion; Life; Ligation; Link; Mediating; Methods; Modeling; Molecular; Monitor; Mutagenesis; Mutation; Myron; National Human Genome Research Institute; Nucleoproteins; Oligonucleotides; Organism; Pathologic Mutagenesis; Pathway interactions; Polymerase; Positioning Attribute; Primer Extension; Process; Prokaryotic Cells; Proteins; Protocols documentation; Rad30 protein; Reaction; Reporter; Reporting; Role; SOS Response; Scientist; Shapes; Single-Stranded DNA; Site; Slide; Son of Sevenless Proteins; Streptavidin; Time; Universities; Uracil; activator 1 protein; base; ds-DNA; flexibility; fluorophore; homologous recombination; human DNA; in vivo; inhibitor/antagonist; miniaturize; repaired; replicase; small molecule; uracil-DNA glycosylase

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). The TLS polymerases gain access to a nascent primer terminus via an interaction with the cells replicative, ring-shaped, clamp (beta-clamp in E.coli and PCNA in eukaryotes). The process is initiated by a clamp loader (gamma-complex in E.coli and replication factor C in eukaryotes), which recognizes the DNA primer terminus and opens and assembles the clamp around the nascent DNA. Each clamp has two (prokaryotes), or three (eukaryotes) potential DNA polymerase binding sites and may, therefore, engage multiple polymerases simultaneously. Indeed, such interactions are believed to be critical for switching between replicative and TLS polymerases. In vitro studies investigating the effects of the replicative clamps on TLS have been hampered because the clamps readily slide off of linear DNA substrates. One option is to cap the DNA ends using large biomolecules such as Streptavidin beads linked to biotinylated oligonucleotides. However, this imposes large steric constraints and may affect the ability of the DNA polymerase to access the primer terminus. Circular, single-stranded templates are, therefore, more likely to provide more informative data on the effects of the replicative clamps on TLS and polymerase switching in vitro. We have therefore developed a protocol for the rapid and efficient purification of circular, single-stranded DNA containing a defined lesion. To achieve our goal, we used a primer containing a site-specific DNA lesion and annealed it to a single-stranded DNA template containing Uracil. After primer extension and ligation, the double-stranded DNA was degraded in vitro using the combined actions of E.coli Uracil DNA glycosylase and Exonucleases I and III. The final product is a circular, single-stranded DNA molecule containing a defined lesion that can be used for in vitro replication and repair assays. 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 RecAATP stoichiometrically from its DNA 3'-end to free pol V (UmuD'2C) to form an active mutasome (pol VMut) with the composition UmuD'CRecAATP. 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 RecAATP in the complex. Reactivation of pol VMut is triggered by replacement of RecAATP from RecA*. Thus, the principal role of RecA* in SOS mutagenesis is to transfer RecAATP to pol V, so as to generate active mutasomal complex for translesion synthesis. Human cells posses at least 14 DNA polymerases (pols). Three, pols alpha, delta and epsilon are involved in genome duplication. The remaining eleven DNA polymerases have specialized functions within the cell. Four of the specialized DNA polymerases (pols eta, iota and kappa and Rev1) belong to the Y-family of DNA polymerases and participate in TLS. Unlike cellular replicases, which are endowed with high processivity, high catalytic efficiency and high fidelity, Y-family TLS DNA polymerases exhibit low processivity, low catalytic efficiency and low fidelity. To facilitate the ongoing studies of the enzymology and cellular roles of these polymerases, a robust and flexible method for monitoring their catalytic activity is needed. In a collaborative study with Anton Simeonovs group (NHGRI), we developed a fluorescence-based assay to study the enzymology of TLS DNA polymerases in real time. The method is based on a fluorescent reporter strand displacement from a tripartite substrate containing a quencher-labeled template strand, an unlabeled primer, and a fluorophore-labeled reporter. With this method, we could follow the activity of human DNA polymerases eta, iota and kappa under different reaction conditions. Last, but not least, we demonstrated that the method can be used for small molecule inhibitor discovery and investigation in highly miniaturized settings and we reported the first nanomolar inhibitors of Y-family DNA polymerases iota and eta. We hypothesize that the fluorogenic replication assays described above should facilitate further mechanistic and inhibitor investigations of the TLS DNA polymerases.

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。 TLS聚合酶通过与细胞复制性环状钳（大肠杆菌中的β-钳和真核生物中的PCNA）的相互作用进入新生引物末端。 该过程由钳加载器（大肠杆菌中的γ-复合物和真核生物中的复制因子C）启动，其识别DNA引物末端并打开和组装新生DNA周围的钳。 每个钳具有两个（原核生物）或三个（真核生物）潜在的DNA聚合酶结合位点，并且因此可以同时接合多个聚合酶。 事实上，这样的相互作用被认为是复制型和TLS聚合酶之间的转换的关键。 研究复制钳对TLS的影响的体外研究受到阻碍，因为钳容易从线性DNA底物上滑落。 一种选择是使用大生物分子（例如与生物素化寡核苷酸连接的链霉抗生物素蛋白珠）来加帽DNA末端。 然而，这施加了大的空间限制，并且可能影响DNA聚合酶接近引物末端的能力。因此，环状单链模板更有可能提供关于复制夹对TLS和聚合酶体外转换的影响的更多信息数据。 因此，我们开发了一种快速有效纯化含有确定病变的环状单链DNA的方案。 为了实现我们的目标，我们使用了含有位点特异性DNA损伤的引物，并将其退火到含有尿嘧啶的单链DNA模板上。 在引物延伸和连接后，使用大肠杆菌尿嘧啶DNA糖基化酶和核酸外切酶I和III的联合作用在体外降解双链DNA。 最终产物是一个环状的单链DNA分子，含有一个确定的病变，可用于体外复制和修复测定。 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RecAATP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'CRecAATP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RecAATP。 通过从RecA* 替换RecAATP来触发pol VMut的再激活。因此，RecA* 在SOS突变中的主要作用是将RecAATP转移到pol V，从而产生用于跨损伤合成的活性突变体复合物。 人类细胞含有至少14种DNA聚合酶（pols）。 第三，pols alpha，delta和pols beta参与基因组复制。 剩下的11种DNA聚合酶在细胞内有专门的功能。四种专门的DNA聚合酶（pols eta、iota和kappa以及Rev 1）属于DNA聚合酶的Y家族并且参与TLS。与具有高合成能力、高催化效率和高保真度的细胞复制酶不同，Y家族TLS DNA聚合酶表现出低合成能力、低催化效率和低保真度。 为了促进这些聚合酶的酶学和细胞作用的正在进行的研究，需要一个强大的和灵活的方法来监测其催化活性。在与Anton Simeonovs小组（NHGRI）的一项合作研究中，我们开发了一种基于荧光的测定方法来真实的研究TLS DNA聚合酶的酶学。该方法是基于从包含淬灭剂标记的模板链、未标记的引物和荧光团标记的报告物的三重底物置换荧光报告物链。利用该方法可以跟踪不同反应条件下人DNA聚合酶eta、iota和kappa的活性。最后，但并非最不重要的是，我们证明了该方法可以用于在高度小型化的环境中发现和研究小分子抑制剂，并且我们报道了Y家族DNA聚合酶iota和eta的第一个纳摩尔抑制剂。我们假设，上述荧光复制试验应有助于进一步的TLS DNA聚合酶的机制和抑制剂的调查。