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

基因组完整性实验室（LGI）的科学家研究了将突变引入DNA的机制。 这些研究跨越了进化光谱，包括细菌、古生菌和真核生物的研究 大肠杆菌中的大多数损伤诱导的诱变依赖于UmuD ′ 2C蛋白复合物，其包含DNA聚合酶V（pol V）。 pol V的生物化学表征受到这样的事实的阻碍，即该酶是众所周知的难以纯化，主要是因为过量产生的UmuC是不溶性的。 在过去的一年中，我们报道了一种简单而有效的协议，用于从几升细菌培养物中快速纯化毫克量的pol V。 不是过量产生UmuC蛋白，而是以低基础水平表达，而UmuD '2从具有诱导型启动子的高拷贝数质粒反式表达。 基于我们在纯化大肠杆菌UmuC方面的成功，我们开发了一系列质粒载体，用于可溶性表达和随后纯化重组蛋白，这些重组蛋白在历史上被证明极难从大肠杆菌中纯化。 类似于UmuC，不是显著过量产生重组人蛋白，而是以低基础水平表达，这有利于重组蛋白的正确折叠并增加其溶解度。因此，传统上难以纯化的高活性重组蛋白很容易使用标准亲和标签和常规色谱法纯化。我们通过从大肠杆菌表达和纯化全长人DNA聚合酶eta、iota和nu证明了这些载体的实用性，并显示纯化的DNA聚合酶在体外具有催化活性。 对高度纯化的大肠杆菌pol V的体外表征表明，在β夹/γ夹加载复合物和反式RecA核蛋白丝（RecA*）存在下，它对SSB包被的环状DNA模板表现出稳健的活性。（在体外形成的由UmuD ′ 2CRecAATP组成的复合物），其被单链结合蛋白募集到引物末端。值得注意的是，在这些条件下，野生型pol V Mut有效地将核糖核苷掺入DNA中。 UmuC的空间门中的Y11 A取代进一步降低了pol V糖的选择性，并有效地将pol V Mut转化为引物依赖性RNA聚合酶，该聚合酶能够以与DNA合成相当的持续合成能力合成长RNA。虽然Y11 F取代对糖选择性的影响最小，但它导致体内自发诱变的增加。相比之下，F10 L取代增加了糖选择性和pol V Mut的整体保真度。 分子模拟分析表明，L10的支链侧链撞击在Y11的苯环上，从而限制其运动，因此，牢固地关闭了空间门，这在野生型酶中不能以足够的严格性防止rNTPs掺入。 我们还分析了三种UmuC空间门突变体（F10 L，Y11 A和Y11 F）在体外促进环丁烷嘧啶二聚体（CPD）的跨损伤DNA合成（TLS），以及促进UV诱导的诱变和体内细胞存活的能力。pol V（UmuC_F10L）突变体比野生型pol V更好地区分rNTP和不正确的dNTP掺入，并且尽管表现出降低的体外绕过CPD的能力，但以高保真度这样做，因此在体内产生最小的UV诱导的诱变。 相比之下，pol V（UmuC_Y11A）在体外CPD的有效TLS期间容易错误掺入rNTP和dNTP两者。 然而，表达umuD ′ C（Y11 A）的细胞比表达野生型umuD ′ C或umuD ′ C（Y11 F）的细胞对紫外线敏感得多，并且表现出较低水平的紫外线诱导的诱变。 我们提出umuD 'C（Y11 A）的UV敏感性增加和UV突变性降低是由于TLS期间rNTPs的过度掺入，其随后被靶向用于修复，而不是不能穿过UV诱导的损伤。 在与Philip Holliger（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聚合酶底物特异性，这对基因组完整性和聚合酶在生物技术中的应用至关重要。我们报告了一个新的特异性检查点位于超过25埃的聚合酶拇指亚域中的活性位点的发现。在Tgo中，来自Thermococcus gorgonarius的复制型DNA聚合酶，我们确定了该区域内的单个突变（E664 K），该突变使跨模板脱碱基位点或环丁烷胸苷二聚体的translesion合成成为可能。结合活性位点中的经典位阻门突变（Y 409 G），E664 K将Tgo DNA聚合酶转化为能够合成长达1.7kb的RNA的RNA聚合酶。我们发现E664 K通过选择性地增加聚合酶对非同源RNA：DNA双链体的亲和力以及降低NTP掺入的Km来实现RNA合成。因此，“看门人”突变鉴定了从DNA到RNA聚合酶的适应性路径中的关键缺失步骤，并定义了聚合酶底物特异性的新的合成后决定因素，其对非同源核酸聚合物的合成和复制具有影响。

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.