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

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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.