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

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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.