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

DNA复制是有压力的，因为复制叉子解开DNA链会产生很高的 必须去除的结构化的超螺旋DNA，否则复制叉将停滞。DNA拓扑异构酶 是普遍存在的酶，对于放松DNA复制过程中形成的超螺旋障碍是必不可少的。 尽管拓扑异构酶如何缓解DNA菌株的机制已经被很好地描述，但这些机制是如何 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，在 复制分叉，仍然知之甚少。作为以拓扑异构酶为靶点的治疗药物 癌症和细菌感染等疾病，更好地了解这些酶将是 未来的药物开发。我的博士后研究发现了一种重要的染色体结构蛋白 在新月形杆菌中称为GapR，它是复制所必需的，特别是 识别过度扭曲的DNA，例如复制分叉之前的DNA。关键的是，GapR还刺激了 细菌拓扑异构酶、旋转酶和拓扑异构酶IV松弛高度超螺旋DNA的活性未知 机制。这项提案将通过研究染色体是如何 结构蛋白如GapR在复制过程中控制拓扑异构酶的活性。目标1将阐明 GapR刺激拓扑异构酶活性的机制。我会进行免疫共沉淀分析 确定GapR是否与拓扑异构酶相互作用以刺激其活性。可替换地或附加地，GapR 可以通过结合过度扭曲的DNA来捕获超级螺旋，从而允许这些被捕获的超级螺旋 通过旋转酶和Topo IV更有效地放松。我将通过研究GapR与DNA的相互作用来测试这一想法 使用磁性镊子。最后，我将使用磁镊子直接评估GapR如何刺激 拓扑异构酶催化循环。目标2将研究GapR如何调节拓扑异构酶的定位和活性 以促进复制。我将确定GapR的缺失如何影响拓扑异构酶与ChIP-Seq和 通过对捕获的DNA进行测序，建立检测全基因组拓扑异构酶活性的方法 具有催化活性的拓扑异构酶。我还将评估GapR的丢失如何影响复制分叉进程， 特别是在具有超螺旋的区域，通过检查复制解旋酶与芯片序列的结合。 在目标3中，我将使用质谱学和 基于转座子的屏幕。我随后将用生化方法鉴定任何已鉴定的辅因子蛋白， 生物物理学和遗传学方法。这些目标内的实验将在K99阶段启动 将包括使用磁镊子进行培训，以研究拓扑异构酶机制和高密度脂蛋白。 通过性方法研究体内拓扑异构酶活性。总而言之，这项提案中的实验将 描述细胞如何使用拓扑异构酶辅助因子蛋白来缓冲DNA过程中产生的压力 复制并确定未来抗菌疗法的潜在靶点。

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.!