Mechanism and Consequences of Temporal Gene Expression for SOS-induced Mutagenesis

SOS 诱导突变的时间基因表达的机制和后果

基本信息

批准号：
10453969
负责人：
MATTHEW J CULYBA
金额：
$ 4.19万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10453969
关键词：
Address Affinity Alleles Antibiotic Resistance Antibiotics Area Bacteria Bacterial DNA Behavior Benefits and Risks Binding Sites Biochemical Biochemistry Communities DNA Damage DNA Repair Gene Data Dose Engineering Enzymes Equilibrium Event Evolution Exposure to Face Gene Expression Genes Genetic Heritability Homeostasis Individual Link Measures Mentors Mutagenesis Mutation Other Genetics Pathway interactions Pennsylvania Positioning Attribute Process Public Health Regulation Research Research Personnel Resistance Route SOS Response Series Site Son of Sevenless Proteins Source Stimulus Stress Structure Synthetic Genes System Techniques Testing Transcription Repressor Universities Variant bacterial genetics biological adaptation to stress career development combat design drug discovery falls fitness genome sequencing genotoxicity innovation new therapeutic target novel drug class novel strategies operation prevent promoter repaired resistance mechanism resistance mutation response synthetic biology whole genome

项目摘要

Project Summary/Abstract We face a public health crisis due to antibiotic resistance, making it imperative to understand how bacteria adapt to antibiotics. The bacterial DNA damage response (SOS response), is a genetic circuit that coordinates the expression of genes linked to the acquisition of resistance. Our data point to a circuit mechanism which enables an extreme separation of error-free and error-prone repair activities at high doses of DNA damage. We believe understanding this mechanism is important, as it may promote the acquisition of antibiotic resistance and thus reveal a novel target for therapy. In this proposal we explore the mechanisms and consequences of temporal gene expression for SOS-induced mutagenesis through the following specific aims: Aim 1. What factors dictate the extent and timing of promoter activity for SOS genes? LexA affinity, SOS gene promoter structure, and the type of DNA damage may all influence the extent and timing of promoter activity within the SOS gene network. We propose to pair biochemistry with a synthetic biology approach to understand how each of these individual factors independently impacts timing in the circuit in order to elucidate the underlying mechanisms responsible for temporal control of promoter activity. Aim 2. What is the mechanism of dose-dependent timing of promoter activities? The lexA promoter, itself, contains binding sites for LexA, placing the SOS-circuit under negative autoregulation. Our data suggest that functional disruption of autoregulation at high doses of DNA damage is critical to achieve the extreme timing differences we observe. We propose to engineer bacterial strains with altered autoregulation of the SOS response to understand how timing of gene expression is achieved. Aim 3. Is the temporal ordering of SOS promoter activities functionally important? Enzymes involved in error-free repair and those involved in error-prone repair may compete for the same damaged DNA substrates. Appropriate timing of these activities may be critical to promote resistance. To test this idea we will engineer bacterial strains with altered timing of these two activities and assess for effects on survival, fitness, and mutational phenomena when exposed to genotoxic antibiotic stress. These studies will uncover new mechanisms for how bacteria adapt to stress and control the timing of gene expression. The information will predict the behavior of other genetic circuits and will inform new approaches in antibiotic drug discovery that aim to suppress mutagenesis in order to prevent the acquisition of antibiotic resistance mutations. It will also extend the PI, who is well versed in biochemical studies, into new areas involving synthetic biology, bacterial genetics, and whole genome sequencing. The combination of a dedicated mentoring team, rigorous plans for career development, and opportunities for integration into a vibrant research community at the University of Pennsylvania will position the PI to become a leading independent researcher dedicated to addressing the problem of antibiotic resistance.

项目摘要/摘要由于抗生素抗性，我们面临公共卫生危机，因此必须了解细菌如何适应抗生素。细菌DNA损伤反应（SOS响应）是一种统计的遗传回路基因的表达与抗药性的获取有关。我们的数据指向电路机构在高剂量的DNA损伤下，实现无错误和容易出错的修复活动的极端分离。我们认为理解这种机制很重要，因为它可能会促进抗生素的获取抗性，因此揭示了一种新的治疗靶标。在此提案中，我们探讨了机制和时间基因表达对SOS诱导的诱变的后果通过以下特定目的：目标1。哪些因素决定了SOS基因的启动子活性的程度和时机？ Lexa Affinity，SOS 基因启动子结构和DNA损伤的类型都可能影响启动子的程度和时机 SOS基因网络中的活动。我们建议将生物化学与合成生物学方法配对了解这些个人因素中的每个因素如何独立影响电路中的时机以阐明负责启动子活动时间控制的基本机制。目标2。启动子活动的剂量依赖时间的机制是什么？ Lexa启动子，本身，包含Lexa的结合位点，将SOS-CIRCUIT放置在负自动调节下。我们的数据暗示高剂量DNA损伤时自动调节的功能破坏对于达到极端是至关重要的我们观察到的时间差异。我们建议通过改变SOS的自动调节来设计细菌菌株回应了解如何实现基因表达的时间。目标3。SOS启动子活动的时间顺序在功能上是否重要？涉及的酶无错误维修和参与易于错误的修复的人可能会竞争相同的受损DNA底物。这些活动的适当时机对于促进抵抗可能至关重要。为了测试这个想法，我们将设计细菌菌株随着这两种活动的时间改变的改变，并评估对生存，健身和突变现象暴露于遗传毒性抗生素应激时。这些研究将发现细菌如何适应压力和控制基因时机的新机制表达。该信息将预测其他遗传回路的行为，并将其告知新方法旨在抑制诱变的抗生素药物发现，以防止获得抗生素抗性突变。它还将将精通生化研究精通的PI扩展到新领域涉及合成生物学，细菌遗传学和整个基因组测序。专用的结合指导团队，严格的职业发展计划以及整合到充满活力的机会宾夕法尼亚大学的研究社区将把PI定位为领先的独立研究人员致力于解决抗生素耐药性问题。