From Comparative Genomics to Comparative Genetics - What is Required for Life Without DNA Replication Origins?

All cells contain a complete copy of the organism's DNA, packaged into chromosomes. Before cells can divide, their chromosomes must be duplicated. This is called DNA replication and begins at specific sites on the chromosome called origins. Bacteria have a single replication origin but organisms with large chromosomes, such as humans, need many origins. We have found that in one case, 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. Haloferax volcanii comes from the Dead Sea, we chose it because the enzymes that carry out DNA replication in archaea are similar to those in eukaryotes, such as human cells.

Haloferax volcanii uses three origins to replicate its chromosome but when all origins are deleted, the cells grow faster. Doing these experiments in humans would be impossible. When origins are deleted from eukaryotes or bacteria, DNA replication is prevented and cells die. So how is Haloferax volcanii able to survive?

Cells without origins use a process called recombination to start DNA replication. Recombination is a form of DNA repair that is used to mend breaks in the chromosome. We found that recombination starts DNA replication at random locations on the chromosome, instead of at specific origins. But if this alternative mode of DNA replication using recombination is more efficient, why have origins at all?

In a sister species of archaea called Haloferax mediterranei, origins cannot be eliminated. When this is attempted, a dormant replication origin becomes active. This means that Haloferax mediterranei needs origins, while Haloferax volcanii can instead use recombination to start DNA replication. Why do these two closely-related microbes behave so differently?

We propose that Haloferax volcanii has critical genes that are missing from Haloferax mediterranei, or vice versa. To simplify the search for these critical genes, we will study the genomes of these and up to 20 additional Haloferax species. Our colleagues in Romania have already discovered that salt lakes in Transylvania are a rich source of Haloferax species. We will test these Haloferax species to see if their origins can be eliminated (as in Haloferax volcanii) or if they are essential (as in Haloferax mediterranei). Then we will compare their genomes to locate the genes responsible.

At the same time, we will examine the consequences of using recombination to start DNA replication.

Haloferax volcanii can use recombination to start DNA replication but this may be hazardous. We will test whether it leads to mutations or chromosome rearrangements, and whether there are alternatives that avoid recombination.

Unlike origins, recombination can take place anywhere on the chromosome, but how often this happens depends on the length of DNA. We will test if there is a minimum size of chromosome for this alternative mode of DNA replication.

Our work will contribute to human health by increasing our understanding of cancer. What we have discovered in Haloferax volcanii has parallels with cancer cells. Haloferax has many copies of its chromosome, this is called polyploidy and helps it to survive when replication and cell division are not coordinated. Cancer cells often have mutations in the genes that control DNA replication and polyploidy is a common feature of cancer. Another consequence of uncoordinated replication is that cancer cells grow faster than ordinary cells. This is similar to the faster growth we observe with origin-less Haloferax volcanii, which use an alternative mode of DNA replication.

DNA replication initiates at origins, which serve as binding sites for initiator proteins that recruit the replicative machinery. Bacteria replicate from single origins while archaea and eukaryotes replicate using multiple origins. Initiation mechanisms that rely on homologous recombination operate in viruses. We have shown that recombination-dependent replication also operates in archaea.

In the archaeon Haloferax volcanii, deletion of all origins or genes encoding initiator proteins leads to the initiation of replication by recombination - strikingly, this also leads to accelerated growth. If recombination alone can efficiently initiate the replication of a cellular genome, what purpose do origins serve and why they have evolved?

Our results contrast with those obtained in Haloferax mediterranei, where origin-dependent replication is strictly required. Deletion of origins from H. mediterranei leads to the activation of a dormant origin. Why is there such a profound difference between two closely-related species? We will use comparative genomics to contrast species of Haloferax that require origins with those that do not. This screen will identify the genes that are required for life without origins.

Our findings will be of significance for DNA replication in all organisms. We expect to uncover genes that act in the restart of stalled replication forks by recombination and underpin genome stability. However, there are pathways for replication restart that avoid recombination - we will determine which genes act in these alternative pathways. Finally, we will test whether there is a minimum DNA size threshold for replication without origins.

We propose that recombination is the ancestral mechanism for initiation of DNA replication, and that origins are a relatively modern innovation. Origin-less Haloferax may be a window into the evolutionary past - it has the potential to show how this ancestral mechanism was displaced by origin-dependent replication.

Who will benefit from this research?
Outcomes from the research will help with our understanding of genome replication and human diseases associated with its deregulation. The healthcare implications of our research will be of potential benefit to a wide range of patient groups, including cancer sufferers. Disruption of DNA replication contributes to genome instability by leading to chromosome breaks and translocations, and genomic regions with few origins are hotspots for rearrangements in cancer. The biomedical implications of the work fit within BBSRC's Strategic Research Priority "Bioscience for Health".
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. DNA processing enzymes found in H. volcanii are adapted to function in high salt, making them of great value to biotechnology companies. Biotechnology implications of the research underpin an area of expertise that will contribute to quality of life enhancement from economic growth, and fit with BBSRC's Strategic Research Priority "Industrial Biotechnology and Bioenergy".

How will they benefit from this research?
The project aims to understand which genes are needed for life without origins. There are striking parallels between origin-less H. volcanii and cancer cells - polyploidy, accelerated growth and an indifference to controls on replication. We anticipate that our results will be informative about DNA replication in all organisms, since the key enzymes are conserved between archaea and humans. Therefore, this project may help uncover new enzymes that are involved in deregulated DNA replication in cancer cells, which would be a step towards improved therapeutic intervention.
Regarding benefits for biotechnology, Dr Loose collaborates with Oxford Nanopore Technologies and this company has supported a BBSRC CASE PhD student in Dr Allers' lab. The project will use Oxford Nanopore MinION technology to sequence novel Haloferax strains and we will communicate with Oxford Nanopore about our research. Novel enzymes we will uncover may have applications in sequencing technologies. Industrial collaborators such as INVISTA Textiles have exploited our expertise in expressing halophilic proteins in H. volcanii. If commercially viable outcomes arise, steps towards exploitation will be taken with commercialisation services at the University of Nottingham.

What will be done to ensure that they have the opportunity to benefit from this research?
In addition to traditional routes of publication, outcomes from this project will be communicated through web pages, social media, the press office of the University of Nottingham, local schools and science discussion groups, and the BBSRC media office. The postdoctoral researcher will make a YouTube vlog about the isolation of novel Haloferax species from Transylvania and their sequencing using Oxford Nanopore MinION technology. Potential future health benefits of the research will be exploited via colleagues from the medical sciences and in partnership with commercialisation services at the University of Nottingham.

Professional development for staff working on the project
The project offers opportunities for the postdoctoral researcher and technician to acquire additional skills. The research will expose both individuals to genetic, genomic and bioinformatic techniques (comparative genomics), with corresponding opportunities for skill development. Before traveling to Transylvania, the PDRA will receive training by Dr Loose in MinION technology. Scientific communication skills of the PDRA will be fostered by presenting the research to academic audiences and the general public (e.g. Nottingham's Café Scientifique). Training will be provided by the University of Nottingham Science Outreach Programme and at a Genetics Society Workshop on 'Communicating Your Science'.