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

项目总结/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）是一种基因电路， 与获得抗性相关的基因的表达。我们的数据指向一个电路机制， 能够在高剂量DNA损伤下将无错和易错修复活动极端分离。 我们相信理解这种机制是重要的，因为它可能促进抗生素的获得。 因此揭示了一种新的治疗靶点。在这一建议中，我们探讨了机制， 通过以下特定目的，研究SOS诱导的诱变的时间基因表达的后果： 目标1.什么因素决定SOS基因启动子活性的程度和时间？莱克萨亲和性，SOS 基因启动子的结构、DNA损伤的类型都可能影响启动子的启动程度和启动时间 SOS基因网络中的活性。我们建议将生物化学与合成生物学方法结合起来， 了解这些单独因素中的每一个如何独立地影响电路中的时序，以便阐明 负责启动子活性的时间控制的潜在机制。 目标2.启动子活性的剂量依赖性定时机制是什么？莱克萨启动子， 本身含有莱克萨的结合位点，使SOS-回路处于负性自动调节下。我们的数据表明 在高剂量DNA损伤下，自动调节的功能中断对于实现极端的 我们观察到的时间差异。我们建议工程改造具有改变的SOS自动调节的细菌菌株， 以了解基因表达的时间是如何实现的。 目标3。SOS启动子活动的时间顺序在功能上重要吗？相关酶 无错误修复和参与易错修复的那些可能竞争相同的受损DNA底物。 这些活动的适当时机可能对促进耐药性至关重要。为了测试这个想法，我们将设计 细菌菌株与改变时间的这两个活动，并评估对生存，健身， 当暴露于遗传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表达时间的新机制。 表情这些信息将预测其他遗传电路的行为，并为研究新方法提供信息。 抗生素药物发现，其目的是抑制诱变以防止抗生素的获得 耐药突变它还将把精通生物化学研究的PI扩展到新的领域 包括合成生物学、细菌遗传学和全基因组测序。一个专门的 指导团队，严格的职业发展计划，以及融入充满活力的 宾夕法尼亚大学的研究社区将使PI成为一个领先的独立研究机构， 致力于解决抗生素耐药性问题的研究人员。

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.