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

项目摘要/摘要 由于抗生素耐药性，我们面临着公共健康危机，因此了解细菌是如何 适应抗生素。细菌DNA损伤反应(SOS反应)，是一种协调 与获得抗性相关的基因的表达。我们的数据指向一种电路机制 在高剂量DNA损伤的情况下，能够将无错误和容易出错的修复活动极端分开。 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 从而揭示了治疗的新靶点。在这项提案中，我们探索了 时间基因表达通过以下特定目的对SOS诱导的突变产生影响： 目的1.什么因素决定了SOS基因启动子活性的范围和时间？LexA亲和力，SOS 基因启动子的结构和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结合起来，以 了解这些单独因素中的每一个如何独立地影响电路中的定时，以便阐明 负责启动子活性的时间控制的潜在机制。 目的2.启动子活性的剂量依赖计时机制是什么？Lexa的发起人， 本身包含LexA的结合位点，使SOS-电路处于负自动调节下。我们的数据显示 在高剂量的DNA损伤下，自我调节的功能中断是达到极端效果的关键 我们观察到时间上的差异。我们建议通过改变SOS的自动调节来设计细菌菌株 反应，以了解基因表达的时间是如何实现的。 目的3.SOS启动子活动的时间顺序在功能上重要吗？参与其中的酶 无错误修复和那些容易出错的修复可能会竞争相同的受损DNA底物。 这些活动的适当时机可能对促进耐药性至关重要。为了测试这一想法，我们将设计 改变这两种活动时间的细菌菌株，并评估对生存、健康和 暴露在基因毒性抗生素应激下时的突变现象。 这些研究将揭示细菌如何适应压力和控制基因时机的新机制。 表情。这些信息将预测其他遗传电路的行为，并将为新的方法提供信息 旨在抑制突变以防止获得抗生素的抗生素药物的发现 抗药性突变。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。结合了一种专门的 指导团队，严谨的职业发展计划，以及融入充满活力的 宾夕法尼亚大学的研究界将使PI成为领先的独立人士 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.