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

Lead Research Organisation: University of Nottingham
Department Name: School of Life Sciences


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.

Technical Summary

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.

Planned Impact

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.


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Description DNA replication initiates at replication origins and must be completed successfully for genome duplication to occur. Replication forks are prone to stalling at DNA lesions and homologous recombination plays a central role in the restart of stalled replication forks. We have previously shown that deletion of all replication origins from the halophilic archaeon Haloferax volcanii results in the initiation of DNA replication by recombination, since the RecA-family recombinase RadA becomes essential. Therefore, H. volcanii provides an excellent model for investigating the role of recombination in (re)starting DNA replication forks. RadB is a recombination mediator and a paralogue of RadA, and we have previously shown that RadB and RadA interact in vivo. Here, we show that RadB interacts with a second protein termed RcrA (Recombination Coupled to Replication in Archaea). RcrA is a non-essential CBS domain protein, only present in archaea, which interacts in vivo with proteins involved in replication and repair. We show that in haloarchaea, radB and rcrA are found in an evolutionarily conserved gene neighbourhood that links DNA replication, homologous recombination, and ubiquitin-like protein modification and turnover. RadB interacts in vivo with another protein encoded by this gene neighbourhood: NdnR (Next Door Neighbour of RadB) is a putative Holliday junction resolvase that belongs to the YqgF protein family. Unlike radB, neither deletion of rcrA nor ndnR affects normal cell growth and DNA repair. However, the combination of radB and ndnR becomes essential in the absence of replication origins, and this synthetic lethality in seen only when both radB and ndnR are deleted. Our results suggest that RadB and NdnR play critical but distinct roles in recombination-dependent DNA replication, and that both pathways of replication initiation depend on RadA.
Exploitation Route The aim of this project is to understand the mechanism of remodelling and restarting stalled replication forks by homologous recombination. How this process is regulated is of fundamental interest for understanding genome replication and human diseases associated with its dysregulation, including cancer, since the key proteins involved in DNA replication are conserved between archaea and humans.
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Title Haloferax volcanii strains and plasmids for determining in-vivo protein:protein interaction assays 
Description A split-GFP protein-interaction assay that uses a salt-stable mGFP2 with modifications that increase protein stability in the hypersaline cytoplasm of H. volcanii. The two N-GFP and C-GFP fragments do not by themselves assemble a fluorescent GFP protein in trans, but are capable of doing so when fused to interacting proteins. The assay published by Winter et al (2018) was adapted for use with DNA repair and replication proteins. See below for URL that described original method. Split-GFP assays were performed as described by Winter et al (2018) with the following modifications. H. volcanii transformants were selected on media containing 0.2 ug/ml Novobiocin (Sigma) and 6 ug/ml Mevinolin (Sigma), and plates were incubated at 45oC for 7-8 days. The presence of both plasmids (NovR and MevR) was confirmed by colony PCR. Split-GFP expressing cells were grown at 37oC to an OD600 of 1.0 and then incubated overnight at 30oC. 2 ml of culture was pelleted, washed twice with 1 ml of 18% SW, resuspended in 500 ul SW and transferred to a 48-well plate. Fluorescence was analysed using GE Healthcare Typhoon (excitation wavelength at 488 nm). 
Type Of Material Cell line 
Year Produced 2019 
Provided To Others? No  
Impact The assay is now routinely used to study the interactions of DNA replication, repair and recombination proteins in Haloferax volcanii 
Title Transcriptome analysis of Haloferax volcanii 
Description RNA-seq dataset of transcriptome analysis of different Haloferax volcanii mutant strains 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact Not yet (data still being analysed) 
Description NSF Grant on Regulatory Systems that Integrate DNA Replication, Recombination and Protein Modification 
Organisation University of Florida
Department Department of Microbiology and Cell Science
Country United States 
Sector Academic/University 
PI Contribution Our research team at the University of Nottingham has developed a tractable genetic system for archaea using Haloferax volcanii. Our group has been pivotal in generating selectable markers, gene knockouts, shuttle plasmids, reporter genes, inducible promoters, protein overexpression systems, and a genome sequence. We use these genetic tools to study replication, recombination and repair. DNA replication. We use genetics and genomics to study DNA replication in H. volcanii. In work published in Nature, we show that deletion of origins leads to accelerated growth.
Collaborator Contribution Dr Julie Maupin-Furlow leads an internationally recognized research team in the Department of Microbiology and Cell Science at the University of Florida. They are focused on understanding the molecular mechanisms that govern the physiology and metabolism of microorganisms from extreme environments. They use a multidisciplinary approach that combines proteomic, biochemical, molecular, genomic, and structural biology techniques to study these extremophiles, including the genetically tractable archaeon Haloferax volcanii. They have made key scientific discoveries that have advanced basic concepts in cell biology, including that archaea catalyze ubiquitin-like modifications that regulate metabolic enzyme activity and protein turnover.
Impact None yet (too early).
Start Year 2016
Description Poster presentation on research findings at annual UK Archaea Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Since 2002, the archaeal community in the UK has held its annual workshop in January. It is convened at a different venues each year but has maintained the same format: an afternoon of talks, a poster session and conference dinner, and a morning of talks. Since 2007, the UK Archaea workshop has been generously supported by the Genetics Society and is the annual meeting of the Archaeal Sectional Interest Group.
The XVIth Annual UK Workshop on Archaea, hosted by Cécile Gubry-Rangin, Aberdeen University, took place at the Sir Duncan Rice Library on the Old Aberdeen University campus. This meeting in 2019 coincided with Aberdeen being awarded the 'Scottish University of the Year, 2019' in the Sunday Times Good University Guide.
The meeting was well attended, attracting a total of 50 attendees from UK laboratories. The presentations (both oral and posters) were nearly exclusively given by early-career scientists (MSc students, postgraduates and postdoctoral investigators). All talks (except the plenary) consisted of 20 min presentation followed by 5 min questions. Students always asked the initial questions for each talk, and this recommendation allowed all students to ask at least one question during the meeting. The research contribution spanned a large breath of archaeal biology themes, including molecular biology, cell cycle, ecology and evolution. Following from last year meeting, there has been a continued increased representation of archaeal ecology and evolution topics, which demonstrates the inclusion of this recent archaeal research theme to strong and well-established archaeal cell biology theme. This meeting gave the opportunity for these communities to meet and discuss current and future scientific research and challenges.
The meeting organisation followed closely the planned organisation, with an afternoon session followed by posters on day one and a morning session on day two. Following refreshments and lunch on arrival, the first session of the meeting saw three talks on archaeal microscopy in Sulfolobus acidocaldarius (Buzz Baum, MRC LMCB, London), on DNA replication in Haloferax volcanii (Rebeca Lever, University of Notthingham) and on archaeal genome evolution (Celine Petitjean, University of Bristol). This first session was ended by a short presentation of the Genetics Society by Jonathan Pettitt (Secretary of the Genetics Society, University of Aberdeen), and he also organised a stand of the Genetics Society during the poster session. After coffee break, the plenary speaker, Professor James McInerney (University of Notthingham) gave an outstanding presentation on archaeal genomics, which inspired several attendees to explore comparative archaeal genomics further in their future investigations.
The poster session was popular with 12 posters on display and was held on Thursday evening coinciding with an evening reception and it generated enthusiastic reflection of the talks, posters and considerable further discussion. The subsequent conference dinner was held in Aberdeen City Centre at Howies restaurant, where the atmosphere was highly favourable to further scientific and collaborative discussion throughout the evening.
Year(s) Of Engagement Activity 2019
Description Public lecture on Archaea at Nottingham Festival of Science and Curiosity, hosted by British Science Association, 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact 40 members of public attended a 20-minute talk on "Archaea and the meaning of life" given at the Bread and Bitter pub in Nottingham, February 2019, which was followed by one hour of lively questions.
Year(s) Of Engagement Activity 2019
Description Public lecture on Archaea at Nottingham SciBar, hosted by British Science Association, 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact 30 members of public attended a 20-minute talk on "Archaea and the meaning of life" given at the Vat & Fiddle pub in Nottingham, September 2018, which was followed by one hour of lively questions.
Year(s) Of Engagement Activity 2018