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

基因组完整性实验室(LGI)的科学家研究将突变引入DNA的机制。这些研究跨越了进化的范围，包括对细菌、古菌和真核生物的研究。 在大肠杆菌中，大多数损伤诱导的突变依赖于UmuD‘2C蛋白复合体，该复合体由DNA聚合酶V(PolV)组成。POL V的生化特性一直受到这样一个事实的阻碍，即众所周知，这种酶很难纯化，很大程度上是因为过量生产的UMUC是不可溶的。在过去的一年里，我们报告了一种简单而有效的方法，可以从几升细菌培养物中快速纯化毫克量的polV。它不是过度生产UMUC蛋白，而是在低基础水平表达，而UmuD‘2是从具有可诱导启动子的高拷贝数质粒反式表达的。在我们成功纯化E.coliUMUC的基础上，我们开发了一系列的重组蛋白的可溶性表达和纯化的质粒载体，这些重组蛋白在历史上被证明是极难从E.Coli中纯化的。与UMUC类似，它没有显著地过度生产重组人蛋白，而是在低基础水平下表达，这有助于重组蛋白的正确折叠并增加其溶解性。因此，传统上难以纯化的高活性重组蛋白可以很容易地使用标准亲和标签和常规层析进行纯化。我们通过从大肠杆菌中表达和纯化全长的人DNA聚合酶Eta、IOTA和Nu，证明了这些载体的实用性，并表明纯化的DNA聚合酶在体外具有催化活性。 对高纯度的E.coliPolV的体外鉴定表明，它在单链断裂包被的环状DNA模板上表现出很强的活性，在β-夹/伽马夹-负载复合体和RecA核蛋白细丝(RecA*)的存在下，反式。这种强大的活性归因于PolV Mut(一种由UmuD‘2CRecAATP组成的体外形成的复合体)出人意料的高处理能力，它被单链结合蛋白招募到引物末端。值得注意的是，在这些条件下，野生型PolV Mut将核糖核苷有效地整合到DNA中。UMUC空间门上的Y11A取代进一步降低了PolV糖的选择性，并有效地将PolV Mut转化为引物依赖的RNA聚合酶，能够合成长RNA，其加工能力与DNA合成相当。虽然Y11F替换对糖的选择性影响很小，但它导致了体内自发突变的增加。相反，F10L替代增加了糖的选择性和PolV Mut的总体保真度。分子模拟分析表明，L10的支链侧链冲击到Y11的苯环上，从而限制了它的运动，从而牢固地关闭了空间门，这在野生型酶中未能足够严格地阻止rNTPs的掺入。 我们还分析了三个UMUC立体门突变体(F10L、Y11A和Y11F)在体外促进环丁烷嘧啶二聚体(CPD)跨损伤DNA合成(TLS)的能力，以及在体内促进紫外线诱变和细胞存活的能力。PolV(UMUC_F10L)突变体对rNTP和错误的dNTP掺入的鉴别能力远远好于野生型polV，虽然在体外表现出绕过CPD的能力降低，但这样做的保真度很高，因此在体内产生了最小的紫外线诱导突变。相反，PolV(UMUC_Y11A)在体外对CPD进行有效的TLS时，很容易错配rNTPs和dNTPs。然而，表达umuD‘C(Y11A)的细胞比表达野生型umuD’C或umuD‘C(Y11F)的细胞对紫外线的敏感性要高得多，并且表现出较低的紫外线诱变水平。我们认为，umuD‘C(Y11A)对紫外线的敏感性增加和紫外线突变性降低是由于在TLS过程中过度掺入rNTP，这些RNTP随后被靶向修复，而不是无法穿越紫外线诱导的损伤。 在与菲利普·霍利格(英国剑桥医学研究理事会)的合作项目中，我们进一步研究了DNA聚合酶底物的特异性，这是基因组完整性和生物技术中聚合酶应用的基础。我们报告了一个新的特异性检查点的发现，该检查点位于聚合酶拇指亚区活性部位的25埃以上。在TGO中，我们发现了该区域内的一个单一突变(E664K)，该突变使跨模板碱基或环丁烷胸苷二聚体的跨损伤合成成为可能。结合活性部位的典型立体门突变(Y409G)，E664K将TGO DNA聚合酶转化为RNA聚合酶，能够合成长达1.7kb的RNA。我们发现E664K通过选择性地增加聚合酶对非同源RNA：DNA双链的亲和力以及降低NTP掺入的Km来使RNA合成。因此，“把关人”突变确定了从DNA到RNA聚合酶的适应途径中一个关键的、缺失的步骤，并定义了一个新的聚合酶底物专一性的合成后决定因素，这对非同源核酸聚合物的合成和复制具有重要意义。

Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into DNA. These studies span the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes Most damage induced mutagenesis in Escherichia coli is dependent upon the UmuD'2C protein complex, which comprises DNA polymerase V (pol V). Biochemical characterization of pol V has been hindered by the fact that the enzyme is notoriously difficult to purify, largely because overproduced UmuC is insoluble. In the past year, we reported a simple and efficient protocol for the rapid purification of milligram quantities of pol V from just a few liters of bacterial culture. Rather than overproducing the UmuC protein, it was instead expressed at low basal levels, while UmuD'2 was expressed in trans from a high copy-number plasmid with an inducible promoter. Based upon our success in purifying E.coli UmuC, we developed a series of plasmid vectors for the soluble expression and subsequent purification of recombinant proteins that have historically proven extremely difficult to purify from E.coli. Similar to UmuC, instead of dramatically overproducing the recombinant human protein, it was instead expressed at a low basal level that facilitates the correct folding of the recombinant protein and increases its solubility. As a consequence, highly active recombinant proteins that are traditionally difficult to purify were readily purified using standard affinity tags and conventional chromatography. We demonstrated the utility of these vectors, by expressing and purifying full-length human DNA polymerases eta, iota and nu, from E.coli and showed that the purified DNA polymerases are catalytically active in vitro. Characterization of highly purified E.coli pol V in vitro revealed that it exhibits robust activity on an SSB-coated circular DNA template in the presence of the beta clamp/gamma clamp-loading complex and a RecA nucleoprotein filament (RecA*) in trans. This strong activity was attributed to the unexpectedly high processivity of pol V Mut (a complex that formed in vitro consisting of UmuD'2CRecAATP), which was recruited to a primer terminus by Single stranded binding protein. Remarkably, under these conditions, wild-type pol V Mut efficiently incorporated ribonucleosides into DNA. A Y11A substitution in the steric gate of UmuC further reduces pol V sugar selectivity and effectively converted pol V Mut into a primer-dependent RNA polymerase that is capable of synthesizing long RNAs with a processivity comparable to that of DNA synthesis. While the Y11F substitution has a minimal effect on sugar selectivity, it resulted in an increase in spontaneous mutagenesis in vivo. In contrast, an F10L substitution increased sugar selectivity and the overall fidelity of pol V Mut. Molecular modeling analysis revealed that the branched side-chain of L10 impinges on the benzene ring of Y11, so as to constrict its movement and as a consequence, firmly closes the steric gate, which in wild-type enzyme fails to guard against rNTPs incorporation with sufficient stringency. We also analyzed the ability of three UmuC steric gate mutants (F10L, Y11A and Y11F) to facilitate translesion DNA synthesis (TLS) of a cyclobutane pyrimidine dimer (CPD) in vitro, and to promote UV-induced mutagenesis and cell survival in vivo. The pol V (UmuC_F10L) mutant discriminated against rNTP and incorrect dNTP incorporation much better than wild-type pol V and although exhibiting a reduced ability to bypass a CPD in vitro, did so with high-fidelity and consequently produced minimal UV-induced mutagenesis in vivo. In contrast, pol V (UmuC_Y11A) readily misincorporated both rNTPs and dNTPs during efficient TLS of the CPD in vitro. However, cells expressing umuD'C(Y11A) were considerably more UV-sensitive and exhibited lower levels of UV-induced mutagenesis than cells expressing wild-type umuD'C or umuD'C(Y11F). We proposed that the increased UV-sensitivity and reduced UV-mutability of umuD'C(Y11A) is due to excessive incorporation of rNTPs during TLS that are subsequently targeted for repair, rather than an inability to traverse UV-induced lesions. In a collaborative project with Philip Holliger (Medical Research Council, Cambridge, UK), we further investigated DNA polymerase substrate specificity, which is fundamental to genome integrity and to polymerase applications in biotechnology. We reported the discovery of a novel specificity checkpoint located over 25 angstroms from the active site in the polymerase thumb sub-domain. In Tgo, the replicative DNA polymerase from Thermococcus gorgonarius, we identified a single mutation (E664K) within this region that enables translesion synthesis across a template abasic site or a cyclobutane thymidine dimer. In conjunction with a classic steric gate mutation (Y409G) in the active site, E664K transforms Tgo DNA polymerase into an RNA polymerase capable of synthesizing RNAs up to 1.7 kb long. We found that E664K enables RNA synthesis by selectively increasing polymerase affinity for the non-cognate RNA:DNA duplex as well as lowering the Km for NTP incorporation. The "gatekeeper" mutation therefore identifies a key, missing step in the adaptive path from DNA to RNA polymerases and defines a novel post-synthetic determinant of polymerase substrate specificity with implications for the synthesis and replication of non-cognate nucleic acid polymers.