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Bilateral NSF/BIO-BBSRC- Remodelling Replication Roadblocks: Regulatory Systems that Integrate DNA Replication, Recombination and Protein Modification

双边 NSF/BIO-BBSRC-重塑复制障碍：整合 DNA 复制、重组和蛋白质修饰的监管系统

基本信息

批准号：
1642283
负责人：
Julie Maupin-Furlow
金额：
$ 40.23万
依托单位：
University of Florida
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-15 至 2020-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1642283&HistoricalAwards=false
关键词：
Bilateral NSF BIO BBSRC Remodelling

项目摘要

In the absence of replication, most DNA lesions (damaged areas of DNA) are tolerated remarkably well by living cells. However, all organisms must replicate (copy) their genome in order to divide, and replication forks are prone to stalling at lesions. Attempting to replicate a DNA lesion will turn a small problem affecting one DNA strand to potentially damaging both DNA strands. Cells can disassemble and rebuild replication forks, which is accomplished by a type of protein degradation. Such remodeling is necessary before the stalled fork can be restarted. In this collaborative project investigators from the US (University of Florida and Duke University) and the UK (Nottingham University) will investigate the regulatory networks that connect the processes of DNA replication, recombination and protein degradation in Archaea. The project provides interdisciplinary training opportunities in quantitative experimental methods. The PIs will also participate in outreach activities to the general public to promote science education.Replication is prone to stalling at DNA lesions but to avoid reinitiating at origins, stalled forks are rebuilt by ubiquitin-mediated protein degradation. Such remodeling is necessary before the stalled fork can be restarted by homologous recombination. The PIs have uncovered a regulatory network centered on the CBS domain protein RcrA in Haloferax volcanii that connects the processes of DNA replication, recombination and ubiquitin-like modification. CBS domain proteins are used as sensors of intracellular metabolites suggesting a regulatory function for RcrA. To unravel this network, a systems biology approach will be used that incorporates transcriptomics, proteomics and two-hybrid analysis. Cells will be challenged by agents that block replication, loss-of-function mutations, and drugs that inhibit key regulatory enzymes. The results will be used to inform computational models, which will be refined using data from iterative rounds of transcriptomics and proteomics.This collaborative US/UK project is supported by the US National Science Foundation and the UK Biotechnology and Biological Sciences Research Council.

在没有复制的情况下，大多数DNA损伤（DNA的受损区域）被活细胞非常好地耐受。然而，所有生物体都必须复制（拷贝）它们的基因组才能分裂，复制叉容易在病变处停滞。试图复制DNA损伤将把影响一条DNA链的小问题变成可能损害两条DNA链的问题。细胞可以分解和重建复制叉，这是通过一种蛋白质降解完成的。在停止的分叉可以重新启动之前，这样的重构是必要的。在这个合作项目中，来自美国（佛罗里达大学和杜克大学）和英国（诺丁汉大学）的研究人员将研究连接DNA复制，重组和蛋白质降解过程的调控网络。该项目提供了定量实验方法的跨学科培训机会。研究员亦会参与外展活动，向市民推广科学教育。复制过程中，DNA会受到损伤，复制过程会停顿下来，但为了避免复制过程重新开始，复制过程中停顿下来的分叉会通过泛素介导的蛋白质降解来重建。在停滞的分叉可以通过同源重组重新启动之前，这种重塑是必要的。PI发现了一个以Haloferax volcanii中CBS结构域蛋白RcrA为中心的调控网络，该网络连接DNA复制，重组和泛素样修饰的过程。CBS结构域蛋白被用作细胞内代谢物的传感器，表明RcrA的调节功能。为了解开这个网络，将使用系统生物学方法，包括转录组学，蛋白质组学和双杂交分析。细胞将受到阻断复制、功能丧失突变和抑制关键调节酶的药物的挑战。研究结果将被用于为计算模型提供信息，这些模型将使用转录组学和蛋白质组学迭代轮的数据进行完善。这一美英合作项目得到了美国国家科学基金会和英国生物技术和生物科学研究理事会的支持。