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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

基本信息

批准号：
10908165
负责人：
ROGER WOODGATE
金额：
$ 232.54万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/10908165
关键词：
Academy Active Sites Antibiotic Resistance Antibiotic susceptibility Antibiotics Antitubercular Agents Archaea Australia Azoles Bacillus Bacteria Binding Biochemical Biological Assay Boronic Acids C-terminal Chromosomes Closure by clamp Collaborations Complex DNA DNA Damage DNA Polymerase III DNA Repair DNA biosynthesis DNA cassette DNA lesion DNA polymerase V DNA replicase DNA-Directed DNA Polymerase Docking Drug resistant Mycobacteria Tuberculosis Escherichia coli Eukaryota Eukaryotic Cell Evolution Excision Excision Repair Family Genetic Genomic DNA Genotoxic Stress Genus Mycobacterium Holoenzymes Human Hydrophobicity International LacZ Genes Libraries Mediating Methods Molecular Mutagenesis Mutation Mutation Spectra Nitriles Nucleotide Excision Repair Organism Pathway interactions Phenotype Play Poland Polishes Polymerase Process Prokaryotic Cells Property Proteins Proteolysis Publishing Queensland Reaction Research Resistance Ribonucleases Ribonucleotides Role SOS Response Science Scientist Serine Site Social Impacts South Africa Specificity System Technology Testing Therapeutic Transcription Repressor Universities acquired drug resistance base design economic impact gel electrophoresis genome-wide in vivo inhibitor lead optimization mimetics molecular modeling mutant mycobacterial novel operation pharmacologic preclinical trial prevent repaired replicase response ribonuclease H1 small molecule tuberculosis chemotherapy tuberculosis drugs

项目摘要

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.

DNA复制、修复和突变（SDRRM）部分的科学家研究了将突变引入DNA的机制。这些研究传统上跨越了进化谱，包括细菌，古生菌和真核生物的研究，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的两条链的复制由单个复制酶DNA聚合酶III全酶（pol III HE）进行。然而，在某些遗传背景下，低保真度TLS聚合酶，DNA聚合酶V（pol V）获得未受损的基因组DNA，其中它促进优先在滞后链上的自发诱变水平升高。作为与位于波兰华沙的波兰科学院的科学家合作的一部分，我们使用pol III（pol III alpha_S759 N）和pol V（pol V_Y11 A）的活性位点突变体来分析核糖核苷酸从E. coli染色体在全基因组范围内的正常复制条件下，以及SOS诱导。使用各种方法来调整这些聚合酶的特定性质（例如，lacI突变谱分析、lacZ回复突变试验、HydEn-seq和碱性凝胶电泳），我们提出了证据，证明在E.大肠杆菌是不相等的。虽然RNA酶HII在前导链核糖核苷酸切除修复（RER）中起主要作用，但滞后链受到其他修复系统（RNA酶HI和SOS激活条件下的核苷酸切除修复）的影响。重要的是，我们认为RNase HI活性也可以影响由复制酶pol III HE掺入滞后链的单个核糖核苷酸的修复。莱克萨酶裂解抑制剂的鉴定随着抗生素耐药性越来越普遍，社会和经济影响越来越紧迫。事实上，细菌已经发展了SOS反应，这有助于在遗传毒性胁迫下进化抗性。转录抑制因子莱克萨在这种反应中起着关键作用。将莱克萨突变为防止诱导SOS反应的不可裂解形式使细菌对抗生素敏感。用小分子实现对蛋白水解的相同抑制也增加抗生素敏感性并减少耐药性的获得。以前开发抑制剂的尝试已经研究了与疏水裂缝结合的1，2，3-三唑分子和与Ser-119共价结合的硼酸。这些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的昆士兰州理工学院的科学家合作，我们发现莱克萨蛋白的切割位点区域（CSR）是一个经典的II型β-转角，并且已发表的1，2，3-三唑化合物模拟了β-转角。在此基础上，我们采取了双重的方法来鉴定一种新的蛋白水解抑制剂。使用FlexX和CovDock中的分子建模方法将通用共价分子文库和α-转角模拟文库对接到莱克萨C-末端结构域。筛选133个得分最高的分子在碱性条件下抑制莱克萨裂解的能力，然后使用RecA介导的计数器测定法测试最高分子。这项研究导致发现了一种亲电丝氨酸弹头，它可以抑制莱克萨蛋白水解，通过腈部分与Ser-119反应。因此，我们的研究提出了一个起点，击中铅优化，这可能会导致抑制SOS反应，并防止获得抗生素耐药性。分枝杆菌突变体的表征 DNA损伤诱导的诱变基因盒与结核分枝杆菌在抗结核（TB）化疗过程中出现耐药性有关。然而，最低限度地包含DnaE 2聚合酶和ImuA和ImuB辅助蛋白的编码的分枝杆菌突变体的分子组成和操作仍然难以捉摸。作为南非开普敦大学的迪格比华纳领导的一项大型国际合作的一部分，我们将分枝杆菌暴露于DNA损伤剂，并观察到DnaE 2和ImuB与DNA聚合酶III β亚基（β钳夹）共定位于不同的细胞内病灶。值得注意的是，在含有破坏的β夹结合基序的imuB突变体中突变体的遗传失活消除了ImuB-β夹病灶形成，这是通过用灰霉素处理杆菌和在生物化学测定中重现的表型，其中这种β夹结合抗生素使预先形成的ImuB-β夹复合物崩溃。这些观察结果确立了ImuB-β夹相互作用对分枝杆菌中诱变DNA修复的重要性，并将突变体鉴定为设计用于保护抗TB药物免受新出现的耐药性的预防性治疗的靶标。

项目成果

期刊论文数量（34）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Tracking Escherichia coli DNA polymerase V to the entire genome during the SOS response.

DOI：
10.1016/j.dnarep.2021.103075
发表时间：
2021-05
期刊：
DNA repair
影响因子：
3.8
作者：
Faraz M;Woodgate R;Clausen AR
通讯作者：
Clausen AR

Ubiquitin and Ubiquitin-Like Proteins Are Essential Regulators of DNA Damage Bypass.

DOI：
10.3390/cancers12102848
发表时间：
2020-10-02
期刊：
Cancers
影响因子：
5.2
作者：
Wilkinson NA;Mnuskin KS;Ashton NW;Woodgate R
通讯作者：
Woodgate R

Simple and efficient purification of Escherichia coli DNA polymerase V: cofactor requirements for optimal activity and processivity in vitro.

DOI：
10.1016/j.dnarep.2012.01.012
发表时间：
2012-04-01
期刊：
DNA REPAIR
影响因子：
3.8
作者：
Karata, Kiyonobu;Vaisman, Alexandra;Goodman, Myron F.;Woodgate, Roger
通讯作者：
Woodgate, Roger

Escherichia coli UmuC active site mutants: effects on translesion DNA synthesis, mutagenesis and cell survival.

DOI：
10.1016/j.dnarep.2012.06.005
发表时间：
2012-09-01
期刊：
DNA REPAIR
影响因子：
3.8
作者：
Kuban, Wojciech;Vaisman, Alexandra;McDonald, John P.;Karata, Kiyonobu;Yang, Wei;Goodman, Myron F.;Woodgate, Roger
通讯作者：
Woodgate, Roger

Identification and Characterization of Thermostable Y-Family DNA Polymerases η, ι, κ and Rev1 From a Lower Eukaryote, Thermomyces lanuginosus.