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

• 批准号: 9384879
• 负责人: MATTHEW J CULYBA
• 金额: $ 18.89万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9384879
• 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; 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/Corepressor; 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

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.

项目概要/摘要 由于抗生素耐药性，我们面临着公共卫生危机，因此必须了解细菌如何 适应抗生素。细菌 DNA 损伤反应（SOS 反应）是协调的遗传回路 与获得抗性相关的基因表达。我们的数据指出了一种电路机制 能够在高剂量 DNA 损伤下将无错修复活动和易错修复活动完全分离。 我们认为了解这一机制很重要，因为它可能会促进抗生素的获得 耐药性，从而揭示了新的治疗靶点。在本提案中，我们探讨了机制和 SOS 诱导突变的时间基因表达的后果通过以下具体目标： 目标 1. 哪些因素决定 SOS 基因启动子活性的程度和时间？ LexA亲和力，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间 SOS 基因网络内的活动。我们建议将生物化学与合成生物学方法结合起来 了解这些单独因素如何独立影响电路中的时序，以便阐明 负责启动子活性时间控制的潜在机制。 目标 2. 启动子活性的剂量依赖性时序机制是什么？ lexA启动子， 它本身包含 LexA 的结合位点，将 SOS 电路置于负自动调节之下。我们的数据表明 高剂量 DNA 损伤时自动调节功能的破坏对于实现极端的效果至关重要 我们观察到的时间差异。我们建议通过改变 SOS 的自动调节来改造细菌菌株 反应以了解基因表达的时间是如何实现的。 目标 3. SOS 启动子活性的时间顺序在功能上重要吗？参与的酶 无错修复和易错修复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力可能至关重要。为了测试这个想法，我们将设计 改变这两项活动时间的细菌菌株，并评估其对生存、健康和健康的影响 暴露于基因毒性抗生素应激时的突变现象。 这些研究将揭示细菌如何适应压力和控制基因时间的新机制 表达。这些信息将预测其他遗传回路的行为，并将为新方法提供信息 旨在抑制诱变以防止获得抗生素的抗生素药物发现 抗性突变。它还将把精通生化研究的PI扩展到新的领域 涉及合成生物学、细菌遗传学和全基因组测序。专用的组合 指导团队、严格的职业发展计划以及融入充满活力的机会 宾夕法尼亚大学的研究社区将把 PI 定位为领先的独立专家 致力于解决抗生素耐药性问题的研究人员。