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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

基本信息

批准号：
10379402
负责人：
Monica S. Guo
金额：
$ 24.9万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-16 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10379402
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 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 rRNA Operon therapeutic target

项目摘要

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复制是有压力的，因为DNA链被复制叉解绕产生了很高的能量