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

Reference	BB/N016491/1
Principal Investigator / Supervisor	Professor Thorsten Allers
Co-Investigators / Co-Supervisors	Professor Matthew Loose
Institution	University of Nottingham
Department	School of Life Sciences
Funding type	Research
Value (£)	398,053
Status	Completed
Type	Research Grant
Start date	01/08/2016
End date	07/03/2020
Duration	43 months

Abstract

DNA replication is the most fundamental task that cells perform and is initiated at specific sites called origins. Replication is prone to stalling at DNA lesions and to avoid reinitiating at origins, stalled forks are restarted by homologous recombination. However, uncontrolled recombination can lead to genome rearrangements. To avoid this problem, ubiquitin-like modification of the replisome plays a key role in regulating the restart of replication. In eukaryotes, this is driven by the Cdc48 ATPase, which targets ubiquitylated proteins for destruction by the proteasome. We have uncovered a regulatory network in the archaeon Haloferax volcanii that connects the processes of DNA replication, recombination and ubiquitin-like protein modification. To unravel this network, we will use a systems biology approach. A combination of transcriptomics, proteomics and two-hybrid analysis will be used to establish gene interactions. Cells will then 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. Similar regulatory systems have been studied in other model systems but H. volcanii offers a unique advantage: in origin-less strains, recombination is used constitutively to initiate all DNA replication. This stripped-down system is an ideal model to investigate how the restart of replication forks by homologous recombination is regulated. We will use a systems biology approach that combines high-throughput transcriptomic and proteomic methods with genetics, genomics and biochemistry to investigate the links between replication, recombination and ubiquitin-like protein modification.

Summary

Before cells can divide, their chromosomes must be duplicated. This process is called DNA replication and it begins at specific locations on the chromosome called replication origins. Bacteria have a single replication origin but organisms with large chromosomes, such as humans, need many origins. We have found that origins are unnecessary, and that cells without them can grow faster than normal. Our research on DNA replication was carried out in Haloferax volcanii, a member of the archaea. The tree of life is split into three groups: eukaryotes, bacteria and archaea. Archaea are microbes renowned for living in extreme conditions such as acid pools and salt lakes. Haloferax volcanii comes from the Dead Sea, we chose it because the enzymes that carry out DNA replication in archaea are similar to those used in eukaryotes. Haloferax volcanii cells without origins use an alternative method called recombination to start DNA replication. Recombination is a form of DNA repair, it is used to mend breaks in the chromosome. These breaks can arise when DNA replication is stalled, which happens if the DNA is damaged and cannot be duplicated. In fact, recombination is used to restart stalled DNA replication in all organisms, and this may be its primary function. Before recombination can be used to restart stalled DNA replication, the enzymes that are being used to duplicate the DNA must first be removed, so that recombination enzymes can take their place. The disassembly of these enzyme complexes is carried out by a system that tags the proteins with a molecule called ubiquitin - this tag identifies the proteins that are destined for remodelling or destruction. We have identified in Haloferax volcanii a network of enzymes that act in DNA replication, recombination and protein destruction. We will carry out a systematic search for other enzymes that belong to this network. Our goal is to uncover the regulatory mechanism behind the cell's response to stalled DNA replication. To do this, we will use our experimental data to create a computer model of the regulatory system. In turn, this computer model will tell us which genes and proteins are key to the process, and that we should examine in detail. This experimental approach is called systems biology. By using Haloferax volcanii cells without origins, we can ensure that all DNA replication is started by recombination. In complex organisms such as humans, origins have become integrated with cellular processes and it is impossible to delete them without detrimental effects. Therefore, our simplified system will allow us to examine the regulatory mechanisms in detail.

Impact Summary

Who will benefit from this research? The outcomes of the proposed work will help with our understanding of genome replication and the human diseases associated with its dysregulation, including cancer. Genomic regions associated with replication fork stalling are hotspots for rearrangements in cancer, while proteasome inhibitors and drugs that modulate ubiquitylation are used in cancer chemotherapy. Biomedical implications of this work fit within the BBSRC's Strategic Research Priority 3 ("Bioscience for health") and NSF Division of Molecular and Cellular Biosciences (MCB) priority "to promote understanding of complex living systems at the molecular, subcellular, and cellular levels". The proposed work has implications for industrial biotechnology. Haloferax volcanii originates from the Dead Sea and it maintains an osmotic balance with its environment by accumulating molar salt concentrations in its cytoplasm. Enzymes from H. volcanii are adapted to function in high salt and this makes them of great value to biotechnology companies. Biotechnology implications of this work fit within the BBSRC's Strategic Research Priority 2 "Bioenergy and industrial biotechnology" and NSF priorities in the Catalysis and Biocatalysis program. How will they benefit from this research? The project aims to understand how DNA replication fork restart by homologous recombination is modulated by ubiquitin-like protein modification. There are striking parallels between origin-less H. volcanii and cancer cells - polyploidy, accelerated growth and unregulated replication. We anticipate that our results will be informative about the restart of stalled replication forks in humans, since the key enzymes involved in replication are conserved between archaea and humans. Therefore, this project could help uncover new enzymes that are involved in unregulated DNA replication in cancer cells - this would be a step towards improved therapeutic intervention. Regarding the potential for industrial biotechnology, Drs Allers and Loose already collaborate with Oxford Nanopore Technologies Ltd. The new enzymes we will uncover could include DNA polymerases, nucleases and helicases, which have numerous applications in DNA sequencing technologies. Industrial collaborators such as INVISTA Textiles Ltd have been keen to exploit our expertise in expressing halophilic proteins in H. volcanii. If commercially viable outcomes arise, steps towards exploitation will be taken in partnership with commercialisation services at the Universities of Nottingham and Florida. What will be done to ensure that they have the opportunity to benefit from this research? In addition to traditional routes of publication, the outcomes from this project will be communicated through our web pages, social media, the press offices of the Universities of Nottingham and Florida, local schools and science discussion groups, and the BBSRC and NSF media offices. Potential future health benefits will be exploited via colleagues from the medical sciences and in partnership with commercialisation services at the Universities of Nottingham and Florida. Professional development for staff working on the project The project offers many opportunities for the postdoctoral researcher and graduate student to acquire new skills. The collaborative nature of the research will expose both individuals to biochemical, genetic and genomic techniques. Training in proteomics, flow cytometry and microscopy will be provided. Scientific communication skills will be fostered by presenting the research at seminars, conferences, and to the public. Appropriate training will be provided by science outreach programmes, at a Genetics Society Workshop on 'Communicating Your Science' and through the UF Career Resource Centre. Training for the researcher and student will be coupled with effective careers advice so that the benefits to the UK and USA can be maximized.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Microbiology, Systems Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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