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）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。 TLS聚合酶通过与细胞复制，环形，夹具（E.Coli中的β-粘液和真核生物中的PCNA）的相互作用来访问新生的引物末端。 该过程是由夹具加载器（E.Coli中的γ-复合物和真核生物中的复制因子C）启动的，该夹子识别DNA引物末端，并打开并在新生的DNA周围打开并组装夹具。 每个夹具具有两个（原核生物）或三个（真核生物）潜在的DNA聚合酶结合位点，因此可能同时参与多个聚合酶。 实际上，这种相互作用被认为对于在复制和TLS聚合酶之间切换至关重要。 由于夹具很容易从线性DNA底物滑落，因此在研究复制夹子对TLS的影响的体外研究受到了阻碍。 一种选择是使用大型生物分子（例如与生物素化寡核苷酸相关的链霉亲和蛋白珠）限制DNA末端。 但是，这会施加较大的空间约束，并可能影响DNA聚合酶进入底端的能力。因此，圆形的单链模板更有可能提供有关复制夹在体外对TLS和聚合酶切换的影响的更多信息数据。 因此，我们已经开发了一种方案，用于快速有效地纯化含有定义病变的圆形单链DNA。 为了实现我们的目标，我们使用了含有特定位点DNA病变的底漆，并将其退火到包含尿嘧啶的单链DNA模板。 引物延伸和连接后，使用大肠杆菌尿嘧啶DNA糖基酶和外核酶I和III的联合作用在体外降解双链DNA。 最终产物是一种圆形的单链DNA分子，其中包含定义的病变，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Recaatp从其DNA 3'-End转移到Free v（Umud'2c），形成一个活性型突变体（pol vmut），并用组成Umud'crecaatp。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，停用效率更慢，同时保留在复合物中的RECAATP。 Pol Vmut的重新激活是通过从RecA*替换RecAATP触发的。因此，RECA*在SOS诱变中的主要作用是将RECAATP转移到Pol V中，以便生成活跃的型突击型复合物以进行跨性别合成。 人类细胞具有至少14个DNA聚合酶（POLS）。 第三，pols alpha，delta和epsilon参与了基因组重复。 其余11个DNA聚合酶在细胞内具有专门的功能。四种专门的DNA聚合酶（POLS ETA，IOTA和KAPPA和REV1）属于DNA聚合酶的Y家庭，并参与TLS。与细胞复制酶不同，这些复制酶具有较高的加工性，高催化效率和高保真度，Y-家庭TLS DNA聚合酶表现出较低的加工性，低催化效率和低忠诚度。 为了促进对这些聚合酶的酶学和细胞作用的持续研究，需要一种稳健而灵活的方法来监测其催化活性。在与Anton Simeonovs组（NHGRI）的合作研究中，我们开发了一种基于荧光的测定法，以实时研究TLS DNA聚合酶的酶学。该方法基于荧光报告基链的位移，该荧光链位移来自包含淬灭剂标记的模板链，未标记的底漆和荧光团标记的报告基因的三方基材。使用这种方法，我们可以遵循不同反应条件下人类DNA聚合酶ETA，IOTA和KAPPA的活性。最后但并非最不重要的一点是，我们证明了该方法可用于高度微型化的环境中的小分子抑制剂发现和研究，我们报道了Y-家庭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测定法应促进对TLS DNA聚合酶的进一步机械和抑制剂研究。