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

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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.