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

DNA复制是有压力的，因为DNA链通过复制叉解旋产生高度的 结构化的超螺旋DNA必须被移除，否则复制叉将停止。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屏障至关重要。 虽然拓扑异构酶如何缓解DNA应变的机制已经得到了很好的描述，但这些机制如何缓解DNA应变的机制仍然是未知的。 酶在体内的调节以及它们如何定位于基因组的特定区域，例如，领先于 复制分叉，仍然知之甚少。作为靶向拓扑异构酶的治疗剂， 癌症和细菌感染等疾病，更好地了解这些酶将是关键， 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 在新月柄杆菌中被称为GapR，它是复制所必需的， 识别过度扭曲的DNA，例如复制叉之前的DNA。关键的是，GapR还刺激了 细菌拓扑异构酶，促旋酶和拓扑异构酶IV的活性，通过一种未知的方式松弛高度超螺旋DNA 机制这项提案将通过调查染色体如何影响基因组来研究新的监管模式。 结构蛋白如GapR在复制过程中控制拓扑异构酶活性。目标1将阐明 GapR刺激拓扑异构酶活性的机制。我将进行免疫共沉淀试验， 确定GapR是否与拓扑异构酶相互作用以刺激其活性。可替代地或另外地，GapR 可以通过结合过度扭曲的DNA来捕获超螺旋，从而使这些被捕获的超螺旋 通过促旋酶和拓扑异构酶IV更有效地放松。我将通过研究GapR如何与DNA相互作用来验证这一想法 使用磁性镊子。最后，我将使用磁镊直接评估GapR如何刺激神经元。 拓扑异构酶催化循环目的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的结合， 开发检测方法，通过对DNA进行测序，检测全基因组范围内的拓扑异构酶活性， 催化活性拓扑异构酶。我还将评估GapR的缺失如何影响复制叉的进展， 特别是在具有超超螺旋的区域中，通过检查复制解旋酶与ChIP-seq的结合。 在目标3中，我将使用质谱法寻找额外的GapR样拓扑异构酶辅因子， 转座子筛选随后，我将用生物化学， 生物物理学和遗传学方法。这些目标范围内的实验将在K99阶段开始 该奖项，将包括培训与磁镊子研究拓扑异构酶机制和高- 通量方法来研究体内拓扑异构酶活性。总之，本提案中的实验将 描述了细胞如何使用拓扑异构酶辅因子蛋白来缓冲DNA扩增过程中产生的压力， 复制并确定未来抗菌治疗的潜在靶点。

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.!