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

基因组完整性实验室(LGI)的科学家研究突变引入受损DNA的机制。现在已经知道，许多长期参与突变过程的蛋白质实际上是低保真的DNA聚合酶，可以在一个被称为跨病变DNA合成(TLS)的过程中穿过受损的DNA。 TLS聚合酶通过与细胞相互作用而进入新生的引物末端，复制的环状钳(在大肠杆菌中为β-钳，在真核生物中为增殖细胞核抗原)。这个过程是由一个夹子加载器(在大肠杆菌中是伽马复合体，在真核生物中是复制因子C)启动的，它识别DNA引物末端，打开并在新生的DNA周围组装夹子。每个钳子有两个(原核生物)或三个(真核生物)潜在的DNA聚合酶结合位点，因此可以同时与多个聚合酶结合。事实上，这种相互作用被认为是在复制聚合酶和TLS聚合酶之间切换的关键。在体外，研究复制钳子对TLS的影响的研究一直受到阻碍，因为钳子很容易从线性DNA底物上滑动。一种选择是使用大的生物分子来封顶DNA末端，例如连接到生物素化寡核苷酸的链霉亲和素珠子。然而，这施加了很大的空间限制，并可能影响DNA聚合酶接近引物末端的能力。因此，圆形单链模板更有可能提供更多关于复制钳子对TLS和体外聚合酶开关的影响的信息数据。因此，我们开发了一种快速有效地纯化含有明确损伤的环状单链DNA的方案。为了实现我们的目标，我们使用了一种含有位点特异性DNA损伤的引物，并将其与含有尿嘧啶的单链DNA模板进行了退火。在引物延伸和连接后，双链DNA在E.coliUracil DNA糖基酶和外切酶I和III的共同作用下被体外降解。最终产物是含有明确损伤的环状单链DNA分子，可用于体外复制和修复分析。 在大肠杆菌中，大多数损伤诱导(SOS)突变发生在DNA聚合酶V被RecA核蛋白细丝(RecA*)激活时，催化TLS。RecA*在同源重组中的生物学功能以及在SOS反应中介导LexA和UmuD切割的生物学功能已经被很好地理解。相反，RecA*在PolV依赖的诱变TLS中的生化作用仍未得到很好的描述。关于RecA*在TLS中作用的建议已经从将UmuD‘C定位在病变附近的引物/模板DNA上，到涉及通过前进的PolV移动模板上的RecA*细丝的动态相互作用，到一种模型，其中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单链DNA上以反式激活TLS的PolV。作为与南加州大学Myron Goodman合作研究的一部分，我们探讨了迄今为止RecA*在polV依赖的SOS突变中的神秘作用。我们证明了RecA*以化学计量学的方式将单个RecAATP从其DNA 3‘端转移到游离的polV(UmuD’2C)上，形成了一个活性突变体(PolVMut)，其组成为UmuD‘CRecAATP。Pol VMut在没有RecA*的情况下催化TLS，并在从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，同时保留在复合体中的RecAATP。重新激活POL VMut是通过替换RecA*中的RecAATP来触发的。因此，RecA*在SOS诱变中的主要作用是将RecAATP转移到polV，从而产生活性的突变复合体用于跨损伤合成。 人类细胞至少有14个DNA聚合酶(Pols)。三是阿尔法、德尔塔和埃西隆参与了基因组复制。剩下的11个DNA聚合酶在细胞内具有特殊的功能。其中四种专门化DNA聚合酶(Pols Eta、IOTA、Kappa和Rev1)属于DNA聚合酶Y家族，参与TLS。与细胞复制酶不同，Y家族TLS DNA聚合酶具有高加工性、高催化效率和高保真度，但具有低加工性、低催化效率和低保真度。为了促进正在进行的这些聚合酶的酶学和细胞作用的研究，需要一种可靠和灵活的方法来监测它们的催化活性。在与Anton Simeonovs团队(NHGRI)的一项合作研究中，我们开发了一种基于荧光的分析方法来实时研究TLS DNA聚合酶的酶学。该方法基于从含有猝灭剂标记的模板链、未标记的引物和荧光团标记的报告分子的三方底物的荧光报告器链置换。利用这种方法，我们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，我们证明了该方法可以在高度小型化的环境中用于小分子抑制剂的发现和研究，并首次报道了Y-家族DNA聚合酶IOTA和ETA的纳米分子抑制剂。我们推测，上述荧光复制分析应该有助于进一步研究TLS DNA聚合酶的机制和抑制物。

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.