Control of topoisomerase activity during DNA replication by bacterial chromosome structuring proteins

细菌染色体结构蛋白在 DNA 复制过程中控制拓扑异构酶活性

• 批准号: 9805617
• 负责人: Monica S. Guo
• 金额: $ 10万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-16 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9805617
• 关键词: Affect; Award; Bacteria; Bacterial Chromosomes; Bacterial Infections; Binding; Biochemical; Biological Assay; Buffers; Caulobacter; Caulobacter crescentus; Cell Death; Cells; ChIP-seq; Chromosome Structures; Chromosomes; Clinic; Co-Immunoprecipitations; Coupled; DNA; DNA Binding; DNA Sequence; DNA Structure; DNA Topoisomerase IV; DNA Topoisomerases; DNA biosynthesis; DNA replication fork; DNA sequencing; DNA-Directed DNA Polymerase; Disease; Ensure; Enzymes; Eukaryota; Future; Genes; Genome; Head; Immunoprecipitation; In Vitro; Light; Magnetism; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Modeling; Movement; Mycobacterium smegmatis; Phase; Play; Polymerase; Proteins; Pseudomonas aeruginosa; Role; Stress; Structural Protein; Structure; Superhelical DNA; Testing; Therapeutic; Time; Topoisomerase; Training; Work; antimicrobial; base; biophysical techniques; clinically relevant; drug development; experimental study; genetic approach; genome-wide; helicase; in vitro activity; in vivo; protein structure; rRNA Operon; therapeutic target

DNA replication is stressful because unwinding of the DNA strands by the replication fork generates highly structured, supercoiled DNA that must be removed or else the replication fork will stall. DNA topoisomerases are ubiquitous enzymes that are essential for relaxing the supercoiling barriers formed during DNA replication. Although the mechanisms of how topoisomerases relieve DNA strain have been well-described, how these enzymes are regulated in vivo and how they are localized to specific regions of the genome, e.g., ahead of replication forks, remains poorly understood. As therapeutics that target topoisomerases are used to treat disorders such as cancer and bacterial infections, a better understanding of these enzymes will be key for future drug development. My postdoctoral studies identified an essential chromosome structuring protein called GapR in the bacterium Caulobacter crescentus that is required for replication and specifically recognizes overtwisted DNA, such as the DNA ahead of replication forks. Critically, GapR also stimulates the activity of bacterial topoisomerases, gyrase and topo IV, to relax highly supercoiled DNA by an unknown mechanism. This proposal will examine new regulatory paradigms by investigating how chromosome structuring proteins such as GapR control topoisomerase activity during replication. Aim 1 will elucidate the mechanisms by which GapR stimulates topoisomerase activity. I will perform co-immunoprecipitation assays to determine if GapR interacts with topoisomerases to stimulate their activities. Alternatively, or in addition, GapR could trap supercoiling by binding overtwisted DNA and consequently allow these trapped supercoils to be more efficiently relaxed by gyrase and topo IV. I will test this idea by examining how GapR interacts with DNA using magnetic tweezers. Lastly, I will use magnetic tweezers to directly assess how GapR stimulates the topoisomerase catalytic cycle. Aim 2 will examine how GapR regulates topoisomerase localization and activity to promote replication. I will determine how loss of GapR affects topoisomerase binding with ChIP-seq and develop assays to examine topoisomerase activity genome-wide by sequencing the DNA trapped in catalytically active topoisomerases. I will also assess how loss of GapR affects replication fork progression, particularly in regions that have hyper supercoiling, by examining binding of replicative helicase with ChIP-seq. In Aim 3, I will search for additional, GapR-like topoisomerase co-factors using mass spectrometry and transposon-based screens. I will subsequently characterize any identified co-factor proteins with biochemical, biophysical, and genetic approaches. The experiments within these aims will be initiated during the K99 phase of the award and will include training with magnetic tweezers to study topoisomerase mechanism and high- throughput approaches to study topoisomerase activity in vivo. Together, the experiments in this proposal will describe how cells use topoisomerase co-factor proteins to buffer against the stresses generated during DNA replication and identify potential targets for future antimicrobial therapeutics.!

DNA复制是压力很大 结构化的超螺旋DNA必须去除，否则复制叉将停滞不前。 DNA拓扑异构酶 无处不在的酶是放松在DNA复制过程中形成的超螺旋屏障所必需的酶。 尽管拓扑异构酶如何缓解DNA菌株的机制已经很好地描述了，但如何 酶在体内受到调节，以及它们如何定位到基因组的特定区域，例如 复制叉，仍然很少理解。作为靶向拓扑异构酶的治疗剂用于治疗 癌症和细菌感染等疾病，对这些酶的更好理解将是 未来的药物开发。我的博士后研究确定了必不可少的染色体结构蛋白 在细菌caulobacter crescentus中称为GAPR，该复制需要，特别是 识别出明确的DNA，例如复制前叉前的DNA。至关重要的是，GAPR也刺激了 细菌性拓扑异构酶，回酶和托普IV的活性，通过未知的高度编涂DNA的活性 机制。该建议将通过研究染色体如何检查新的调节范例 在复制过程中结构蛋白质，例如GAPR控制拓扑异构酶活性。 AIM 1将阐明 GAPR刺激拓扑异构酶活性的机制。我将执行共免疫沉淀测定 确定GAPR是否与拓扑异构酶相互作用以刺激其活性。或者，GAPR 可以通过绑定明显的DNA来捕获超串联 通过回旋酶和Topo IV更有效地放松。我将通过检查GAPR如何与DNA相互作用来测试这个想法 使用磁镊子。最后，我将使用磁性镊子直接评估GAPR如何刺激 拓扑异构酶催化循环。 AIM 2将检查GAPR如何调节拓扑异构酶定位和活动 促进复制。我将确定GAPR的损失如何影响拓扑异构酶与Chip-Seq和 开发测定方法，通过测序被困在 催化活性的拓扑异构酶。我还将评估GAPR损失如何影响复制叉进展， 特别是在具有超级统计的地区，通过检查复制性解旋酶与芯片序列的结合。 在AIM 3中，我将使用质谱法和 基于转座子的屏幕。随后，我将以生化， 生物物理和遗传方法。这些目标中的实验将在K99阶段启动 该奖项，包括对磁性镊子进行培训，以研究拓扑异构酶机制和高级 在体内研究拓扑异构酶活性的吞吐量方法。该提案中的实验将在一起 描述细胞如何使用拓扑异构酶co蛋白蛋白来缓冲DNA期间产生的应力 复制并确定未来抗菌疗法的潜在靶标。