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 process is called DNA replication and 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 (Hawkins et al. 2013 Nature 503, 544-7).

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 uses several origins to replicate its chromosome. But when all of these origins are removed, the cells actually grow faster. Doing these experiments in humans would be impossible. When origins are eliminated from eukaryotes or bacteria, it prevents DNA replication and leads to death. So how is Haloferax volcanii able to survive?

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. We found that recombination starts DNA replication at random locations on the chromosome, instead of being restricted to a limited number of origins, and this makes the process faster. But this poses a puzzle: if the alternative process using recombination is more efficient, why have replication origins at all?

We propose that origins in Haloferax volcanii are selfish genes. Selfish origins need not offer any advantage to the host cell, but they increase their own frequency because they have hijacked the DNA replication machinery. Over the course of evolution, host cells have found a way to regulate origins and this has allowed them to coordinate the timing of DNA replication with cell division. In complex organisms such as humans, origins have become integrated with cellular processes and it is impossible to delete them without detrimental effects.

The unusual mode of DNA replication we have discovered in Haloferax volcanii has parallels with cancer. Haloferax volcanii has many copies of its chromosome, this is called polyploidy and helps it to survive when replication and cell division are no longer coordinated. Many cancer cells 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. Such accelerated growth is reminiscent of origin-less Haloferax volcanii, which use an alternative mode of replication to outpace other cells.

Our work on a microbe from the Dead Sea has shown how surprising results can come from testing long-held assumptions in unusual organisms. But it has given us as many questions as answers:
- How does this alternative mechanism of DNA replication work? Does it have negative consequences for the cell?
- Does Haloferax volcanii use it all the time? If not, how is it kept in check by 'normal' replication?
- Above all, why does Haloferax volcanii grow faster without origins, when other cells would die? We believe that two aspects of this organism are key: recombination and polyploidy.

We will use a combination of genetic and biochemical tools that we have developed, to examine the effects of recombination and polyploidy on replication. This work has implications for DNA replication in all organisms - it may contribute to our understanding of how cancer cells evade the checks on replication, and give an insight into how DNA was replicated before the evolution of 'selfish' origins.

DNA replication initiates at origins, which serve as binding sites for initiator proteins that recruit the replicative machinery. Origins differ in number across the three domains, bacteria replicate from single origins while most archaea and all eukaryotes replicate using multiple origins. Initiation mechanisms that rely on recombination operate in viruses. We have shown that recombination-dependent replication operates in archaea, and that it can lead to accelerated growth.

We have identified four chromosomal origins in the archaeon Haloferax volcanii. Deletion of individual origins results in reduced growth but a strain lacking all origins grows faster than wild type. These origin-less cells initiate replication at dispersed sites rather than at discrete origins and have an absolute requirement for the recombinase RadA. Thus, recombination alone can efficiently initiate the replication of an entire cellular genome. This raises the question of what purpose origins serve and why they have evolved.

We suggest that origins are selfish elements that have hijacked the host replication machinery. During evolution, cells have found ways to regulate these 'selfish' origins, enabling them to coordinate DNA replication with cell division. H. volcanii is highly polyploid and has no requirement to coordinate replication with division, but it is vital that each of its chromosomes is identical and this requires efficient recombination.

We hypothesise that a combination of polyploidy and recombination is essential for origin-less DNA replication. The consequences of relying exclusively on recombination-dependent replication will be examined - does this lead to genome instability? The role of polyploidy will be tested - is it a precondition for life without origins? The interplay of recombination-dependent and origin-dependent replication will be dissected. We will test the hypothesis that origins are selfish - do they offer any advantage to the host cell?

Who will benefit from this research?

The proposed work has long-term healthcare implications that will be of potential benefit to a wide range of patient groups, in particular cancer sufferers. Disruption of the regulation of DNA replication contributes to genome instability by leading to chromosome breaks, translocations and aneuploidy. Genomic regions with few active origins are hotspots for rearrangements in cancer. Outcomes from the proposed research will help with our understanding of genome replication and the human diseases associated with its deregulation, including cancer and developmental disorders. Thus, the biomedical implications of this work fit within the BBSRC's Strategic Research Priority 3 "Basic bioscience underpinning 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 in its cytoplasm. Therefore, molecular process in H. volcanii have adapted to function in high salt and this makes the DNA processing enzymes we will characterise of great value to biotechnology companies. Thus, the biotechnology implications of this work fit within the BBSRC's Strategic Research Priority 2 "Bioenergy and industrial biotechnology".

How will they benefit from this research?

The project aims to understand how DNA replication is possible without origins. We will work with the genetically tractable archaeon H. volcanii. 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 the regulation of replication in all organisms - 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 of the proposed work for industrial biotechnology, we have an established collaboration with Oxford Nanopore Technologies Ltd., who support a BBSRC CASE studentship in Dr Allers' laboratory. The new enzymes we will uncover could include DNA polymerases, nucleases and helicases, which have numerous applications in DNA sequencing technologies. If commercially viable outcomes arise, steps towards exploitation will be taken with The University of Nottingham Business Engagement and Innovation Services.

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, the replication origin database (OriDB), the University of Nottingham's Communications and Marketing Unit, Nottingham's Café Scientifique and BioCity, and the BBSRC media office. Potential future health benefits will be exploited via colleagues within the Faculty of Medicine and Health Sciences, in particular those in the Division of Pre-Clinical Oncology. Should the project outcomes warrant additional exposure, we will engage the services of Bulletin Academic, a specialist communications consultancy.

Professional development for staff working on the project

The project offers many opportunities for the postdoctoral researcher and technician to acquire additional skills. The collaborative nature of the research will expose both individuals to biochemical, genetic and genomic techniques. Scientific communication skills of the PDRA will be fostered by presenting our research to academic audiences and the general public (e.g. Nottingham's Café Scientifique or local schools). Appropriate training for both audiences will be provided by the University of Nottingham Science Outreach Programme, and at a Genetics Society Workshop on 'Communicating Your Science'.