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的受损区域)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，复制叉子容易在病变处停滞不前。试图复制DNA损伤将把一个影响一条DNA链的小问题变成潜在地破坏两条DNA链。细胞可以分解和重建复制叉子，这是通过一种蛋白质降解完成的。这样的改造是必要的，然后才能重新启动失速的叉子。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)的研究人员将调查连接古生代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。PIS还将参与面向公众的外展活动，以促进科学教育。复制容易在DNA损伤处停滞，但为了避免在起点重新启动，停滞的叉子会通过泛素介导的蛋白质降解来重建。在停滞不前的叉子可以通过同源重组重新启动之前，这种重塑是必要的。PI已经发现了一个以Haloferax Volcanii中的CBS结构域蛋白RCRA为中心的调控网络，该网络连接了DNA复制、重组和泛素样修饰的过程。CBS结构域蛋白被用作细胞内代谢物的感受器，暗示了对RCRA的调节功能。为了揭开这一网络，将使用系统生物学方法，结合转录组学、蛋白质组学和双杂交分析。细胞将受到阻止复制、功能丧失突变和抑制关键调控酶的药物的挑战。结果将被用于提供计算模型，这些模型将使用反复几轮转录和蛋白质组学的数据进行精炼。这项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。

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.