Origins "R" Us: investigating the role of R-loops in origin-independent DNA replication

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The project will investigate the role of R-loops in origin-less DNA replication in Haloferax volcanii, and the impact of this alternative mode of DNA replication on genome stability.DNA replication is initiated at chromosomal sequences called origins. Replication origins are assumed to be essential, but we have shown that in Haloferax volcanii, a member of the Archaea, life without origins is possible.Archaea are the third domain of life, alongside eukaryotes and bacteria. The machinery for DNA replication is strikingly similar in archaea and eukaryotes. Deletion of all replication origins from the main chromosome of Haloferax volcanii is not only possible but leads to accelerated growth. We have previously shown that initiation of DNA replication in the absence of origins involves homologous recombination, since it depends on the archaeal recombinase RadA. Homologous recombination involves the formation of a three-stranded DNA structure called a D-loop, which is catalysed by RadA.We have now obtained new data showing a role for transcription in replication without origins. The genome of Haloferax volcanii consists of a chromosome with three origins, and three mini-chromosomes with one origin each. All origins on the chromosome can be deleted but the origin on the mini-chromosome pHV3 cannot be deleted. Notably, transcription levels on pHV3 are very low. We have engineered a high-activity promoter onto pHV3 to increase transcription, and have found that the origin can now be deleted. This suggests a role for R-loops.R-loops are three-stranded structures composed of a DNA:RNA hybrid, they are typically formed as a result of abortive transcription. R-loops have the potential to directly prime DNA replication from the 3' OH end of the RNA strand. However, R-loops have also been linked to genome instability via the formation of DNA breaks, which in turn may be used to prime DNA replication via homologous recombination.The initial aim is to determine whether R-loops prime origin-less DNA replication directly, or whether they act indirectly via homologous recombination. This will be tested by determining the requirement for the archaeal recombinase RadA in strains that depend on transcription for origin-less replication. Genomic techniques will be used to determine the sites of R-loops (DRIP-seq) and D-loops (ChIP-seq using tagged RadA), in strains with and without origins.Excessive R-loops lead to genome instability in bacteria and eukaryotes, due to transcription-replication conflicts. We will engineer a strain of Haloferax volcanii to flip the direction of a highly transcribed rrn rRNA operon, so that RNA polymerases collide with DNA replication forks from a nearby origin. Alternatively, convergent rrn operons will be used to investigate transcription-transcription conflicts. Genome rearrangements that result from polymerase collisions will be detected by single-molecule long-read Nanopore sequencing.Finally, we will examine the role of RNA polymerase backtracking in R-loop formation. In bacteria, transcription factors that remove stalled RNA polymerase from the template are required for initiation of DNA replication from R-loops. We will test whether this is the case for Haloferax volcanii, by examining the role of archaeal RNA polymerase inhibitors in R-loop formation.