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

在没有复制的情况下，大多数DNA损伤（DNA受损区域）都能被活细胞很好地耐受。然而，所有生物体都必须复制（复制）它们的基因组才能分裂，而复制分叉容易在病变处停滞。试图复制DNA损伤会把一个影响一条DNA链的小问题变成潜在地损害两条DNA链。细胞可以分解并重建复制叉，这是通过一种蛋白质降解来完成的。这样的改造是必要的之前，停滞的叉子可以重新启动。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）的研究人员将研究古细菌中连接DNA复制、重组和蛋白质降解过程的调控网络。该项目提供定量实验方法的跨学科培训机会。他们也会参与向公众推广科学教育的活动。复制容易在DNA损伤处停滞，但为了避免在起源处重新启动，停滞的分叉通过泛素介导的蛋白质降解来重建。这样的重塑是必要的，在停止叉子可以通过同源重组重新启动。pi发现了一个以火山盐藻CBS结构域蛋白RcrA为中心的调控网络，该网络连接了DNA复制、重组和泛素样修饰过程。CBS结构域蛋白被用作细胞内代谢物的传感器，表明RcrA具有调节功能。为了解开这个网络，将使用结合转录组学，蛋白质组学和双杂交分析的系统生物学方法。细胞将受到阻断复制的药剂、丧失功能的突变和抑制关键调节酶的药物的挑战。这些结果将用于计算模型，这些模型将使用来自转录组学和蛋白质组学的迭代数据进行改进。这个英美合作项目由美国国家科学基金会和英国生物技术和生物科学研究委员会支持。

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.