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

DNA复制、修复和突变部门(SDRRM)的科学家研究将突变引入DNA的机制。这些研究传统上跨越了进化的光谱，包括对细菌、古生物和真核生物的研究，并涉及与世界各地的科学家合作。 大肠杆菌中核糖核苷酸切除修复的链特异性 在大肠杆菌中，基因组DNA的两条链的复制是由单个复制酶DNA聚合酶III全酶(PolIII HE)进行的。然而，在某些遗传背景下，低保真的TLS聚合酶，DNA聚合酶V(PolV)获得了未受损的基因组DNA，在那里它优先促进滞后链上自发突变水平的升高。作为与波兰华沙波兰科学院科学家合作的一部分，我们利用polIII(polIIIα_S759N)和polV(PolV_Y11A)的活性位点突变，在正常复制和SOS诱导的条件下，在全基因组范围内分析了核苷酸的掺入和从大肠杆菌染色体上的移除。使用各种方法调整这些聚合酶的特殊性质(例如，LacI突变谱分析、LacZ逆转试验、Hyden-Seq和碱性凝胶电泳)，我们提出了证据，证明在大肠杆菌中两条DNA链上的核苷酸修复是不相等的。虽然RNase HII在前导链核苷酸切除修复(RER)中起主要作用，但滞后链受到其他修复系统的影响(RNase HI和在SOS激活的条件下也有核苷酸切除修复)。重要的是，我们认为RNase HI活性也可以影响复制酶PolIII HE掺入滞后链的单个核苷酸的修复。 一种LexA裂解抑制剂的鉴定 随着抗生素耐药性变得更加普遍，其社会和经济影响日益紧迫。事实上，细菌已经产生了SOS反应，这有助于在遗传毒性压力下进化抗性。转录抑制因子LexA在这种反应中起着关键作用。LexA突变为不可切割的形式，阻止了SOS反应的诱导，使细菌对抗生素敏感。用小分子实现对蛋白质分解的同样抑制也增加了抗生素的敏感性，减少了耐药性的获得。以前开发抑制剂的尝试已经研究了与疏水裂隙结合的1，2，3-三氮唑分子，以及与Ser-119共价结合的硼酸。这两个结果都没有导致任何分子进入临床前试验。与澳大利亚布里斯班昆士兰理工学院的科学家合作，我们发现LexA蛋白的裂解位点区域(CSR)是一个经典的II型贝塔转弯，而已发表的1，2，3-三唑化合物模仿了贝塔转弯。在此基础上，我们采用双重方法来鉴定一种新的蛋白水解酶抑制剂。使用Flexx和CovDock中的分子模拟方法，将通用共价分子库和a-TURN模拟库对接到Lexa C-末端结构域。对133个得分最高的分子进行筛选，以确定它们在碱性条件下抑制LexA裂解的能力，然后使用RecA介导的反分析测试排名靠前的分子。这项研究导致了一种亲电丝氨酸弹头的发现，这种弹头可以抑制LexA蛋白分解，通过丁腈部分与Ser-119反应。因此，我们的研究为Hit-to-Lead优化提供了一个起点，这可能导致抑制SOS反应并防止获得抗生素耐药性。 分枝杆菌突变酶小体的鉴定 DNA损伤诱导突变基因盒与结核分枝杆菌在抗结核化疗过程中出现耐药性有关。然而，由DNAE2聚合酶和ImuA和ImuB辅助蛋白组成的最低限度编码的分支杆菌突变酶的分子组成和操作仍然难以捉摸。作为南非开普敦大学Digby Warner领导的大型国际合作的一部分，我们将分枝杆菌暴露在DNA损伤剂中，并观察到DNAE2和ImuB与DNA聚合酶IIIβ亚单位(β钳)共同定位于不同的细胞内病灶。值得注意的是，在含有中断的Beta钳结合基序的imuB突变体中，突变酶体的遗传失活取消了ImuB-Beta钳焦点的形成，这是一种通过用格利利霉素处理杆菌而在药理学上概括的表型，在生化分析中，这种Beta钳结合抗生素崩溃了预先形成的ImuB-Beta钳复合体。这些观察证实了ImuB-beta钳相互作用对分枝杆菌突变DNA修复的重要性，并确定突变酶体是旨在保护抗结核药物免受新出现耐药的辅助治疗的靶点。

Scientists in the Section on DNA Replication, Repair and Mutagenesis (SDRRM) study the mechanisms by which mutations are introduced into DNA. These studies have traditionally spanned the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes and involve collaborations with scientists around the world. Strand specificity of Ribonucleotide Excision Repair in E.coli In Escherichia coli, replication of both strands of genomic DNA is carried out by a single replicase DNA polymerase III holoenzyme (pol III HE). However, in certain genetic backgrounds, the low-fidelity TLS polymerase, DNA polymerase V (pol V) gains access to undamaged genomic DNA where it promotes elevated levels of spontaneous mutagenesis preferentially on the lagging strand. As part of a collaboration with scientists at the Polish Academy of Sciences in Warsaw, Poland, we employed active site mutants of pol III (pol III alpha_S759N) and pol V (pol V_Y11A) to analyze ribonucleotide incorporation and removal from the E. coli chromosome on a genome-wide scale under conditions of normal replication, as well as SOS induction. Using a variety of methods tuned to the specific properties of these polymerases (e.g., analysis of lacI mutational spectra, lacZ reversion assay, HydEn-seq, and alkaline gel electrophoresis), we presented evidence that repair of ribonucleotides from both DNA strands in E. coli is unequal. While RNase HII plays a primary role in leading-strand Ribonucleotide Excision Repair (RER), the lagging strand is subject to other repair systems (RNase HI and under conditions of SOS activation also Nucleotide Excision Repair). Importantly, we suggested that RNase HI activity can also influence the repair of single ribonucleotides incorporated by the replicase pol III HE into the lagging strand. Identification of an inhibitor of LexA cleavage As antibiotic resistance has become more prevalent, the social and economic impacts are increasingly pressing. Indeed, bacteria have developed the SOS response which facilitates the evolution of resistance under genotoxic stress. The transcriptional repressor, LexA, plays a key role in this response. Mutation of LexA to a non-cleavable form that prevents the induction of the SOS response sensitizes bacteria to antibiotics. Achieving the same inhibition of proteolysis with small molecules also increases antibiotic susceptibility and reduces drug resistance acquisition. Previous attempts at developing inhibitors have investigated 1,2,3-triazole molecules binding to the hydrophobic cleft, and boronic acids that covalently bound to Ser-119. Neither of these resulted in any molecules going to preclinical trials. In collaboration with scientists at the Queensland Institute of Technology in Brisbane, Australia, we found that the cleavage site region (CSR) of the LexA protein is a classical Type II beta-turn, and that published 1,2,3-triazole compounds mimic the beta-turn. Based upon this, we took a dual approach to the identification of a novel proteolytic inhibitor. Generic covalent molecule libraries and a -turn mimetic library were docked to the LexA C-terminal domain using molecular modelling methods in FlexX and CovDock. The 133 highest scoring molecules were screened for their ability to inhibit LexA cleavage under alkaline conditions and the top molecules were then tested using a RecA-mediated counter assay. This research led to the discovery of an electrophilic serine warhead that can inhibit LexA proteolysis, reacting with Ser-119 via a nitrile moiety. Our studies therefore present a starting point for hit-to-lead optimization, which could lead to inhibition of the SOS response and prevent the acquisition of antibiotic resistance. Characterization of the mycobacterial mutasome A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded mycobacterial mutasome minimally comprising DnaE2 polymerase and ImuA and ImuB accessory proteins remain elusive. As part of a large international collaboration led by Digby Warner at the University of Cape Town, South Africa, we exposure mycobacteria to DNA damaging agents and observed that DnaE2 and ImuB co-localize with the DNA polymerase III beta subunit (beta clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuB mutant containing a disrupted beta clamp-binding motif abolishes ImuB-beta clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this beta clamp-binding antibiotic collapses pre-formed ImuB-beta clamp complexes. These observations established the essentiality of the ImuB-beta clamp interaction for mutagenic DNA repair in mycobacteria and identifies the mutasome as a target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.