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.

关于DNA复制，修复和诱变（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，包括对细菌，古细菌和真核生物的研究，并涉及与世界各地的科学家的合作。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的两条链的复制都是由单个复制酶DNA聚合酶III Holoenzyme（Pol III HE）进行的。但是，在某些遗传背景中，低保真TLS聚合酶DNA聚合酶V（POL V）可获得对未损坏的基因组DNA的访问，在该基因组DNA中，它优先促进了滞后链的自发诱变水平升高。作为波兰华沙科学院的科学家的一部分，我们采用了Pol III（Pol III Alpha_S759N）和Pol V（Pol v_yy11a）的主动现场突变体，分析了核糖核苷酸的融合，并从基因范围尺度上的coli collomosome中删除，并在正常的条件下，以及在常规的条件下，以及SOS的指标。使用多种调谐到这些聚合酶特异性特性的方法（例如，LACI突变光谱，LACZ恢复分析，Hyden-Seq和碱性凝胶电泳），我们提供了证据，证明了E. coli中两个DNA链的核糖核苷酸的修复是不平等的。虽然RNase HII在前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的约束（RNase HI，在SOS激活的条件下，还需要核苷酸切除修复）。重要的是，我们建议RNase HI活性还可以影响复制酶Pol III纳入滞后链的单个核糖核苷酸的修复。鉴定Lexa裂解的抑制剂随着抗生素耐药性变得越来越普遍，社会和经济影响越来越紧迫。实际上，细菌发展了SOS反应，从而促进了遗传毒性应激下抗性的演变。转录阻遏物Lexa在此响应中起着关键作用。 Lexa突变为不可裂解的形式，以防止SOS响应诱导细菌对抗生素的敏感。通过小分子实现蛋白水解的相同抑制作用也会增加抗生素敏感性并降低耐药性的获取。以前的开发抑制剂的尝试已经研究了1,2,3-三唑分子与疏水性裂缝结合，并共价结合Ser-1119。这些都没有导致任何分子进行临床前试验。与澳大利亚布里斯班昆士兰州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）是一种经典的II型Beta-Turn，并且出版了1,2,3-三唑化合物模仿Beta-Turn。基于此，我们采取了双重方法来鉴定一种新型的蛋白水解抑制剂。通用的共价分子库和一个模拟图库使用Flexx和Covdock中的分子建模方法停靠在Lexa C末端结构域。筛选了133个最高评分分子在碱性条件下抑制Lexa裂解的能力，然后使用RECA介导的反分析对顶部分子进行测试。这项研究导致发现了可以抑制Lexa蛋白水解的亲电丝氨酸弹头，并通过硝酸属部分与Ser-119反应。因此，我们的研究提出了命中率优化的起点，这可能导致抑制SOS反应并防止获得抗生素耐药性。分枝杆菌的表征 DNA损伤诱导的诱变基因盒已与抗链球菌（TB）化学疗法期间结核分枝杆菌的耐药性出现有关。然而，编码的分枝杆菌突变体的分子组成和操作最小包含DNAE2聚合酶，IMUA和IMUB辅助蛋白仍然难以捉摸。作为由南非开普敦大学的Digby Warner领导的大型国际合作的一部分，我们暴露于DNA损害代理商，并观察到DNAE2和IMUB与DNA聚合酶III III Beta beta Subunit（Beta夹具）在独特的胞内灶中均与DNA聚合酶III III Beta Subunit（beta夹具）进行了固定。 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.这些观察结果确定了imub-beta夹具相互作用在分枝杆菌中进行诱变DNA修复的重要性，并将其识别为诱变体是旨在保护抗TB药物免受新兴耐药性的辅助治疗剂的靶标。

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.