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

该项目将研究R-环在盐生植物火山II中无起源DNA复制中的作用，以及这种可选的DNA复制模式对基因组稳定性的影响。DNA复制始于被称为起源的染色体序列。复制起源被认为是必不可少的，但我们已经证明，在古生代的一员Haloferax Volcanus II中，可能存在没有起源的生命。古生代是生命的第三个领域，与真核生物和细菌并列。在古生物和真核生物中，DNA复制的机制惊人地相似。删除Haloferax Volcanii主染色体上的所有复制起始点不仅是可能的，而且会导致加速生长。我们以前已经证明，在没有起始点的情况下启动DNA复制涉及同源重组，因为它依赖于古生菌重组酶RADA。同源重组涉及形成一个被称为D-环的三链DNA结构，这是由RADA催化的。我们现在已经获得了新的数据，表明了转录在没有起源的复制中的作用。海参基因组由一个具有三个起源的染色体和三个各有一个起源的小染色体组成。染色体上的所有起始点都可以删除，但微小染色体PHV3上的起始点不能删除。值得注意的是，PHV3上的转录水平非常低。我们已经在PHV3上设计了一个高活性的启动子来增加转录，并发现现在可以删除起始点。这表明了R-环的作用。R-环是由DNA：RNA杂交组成的三链结构，它们通常是由于转录流产而形成的。R-环有可能直接从RNA链的3‘OH端启动DNA复制。然而，R-环也通过DNA断裂的形成与基因组的不稳定性联系在一起，而DNA断裂又可以通过同源重组来启动DNA复制。最初的目的是确定R-环是直接启动无起点的DNA复制，还是通过同源重组间接作用。这将通过确定依赖转录进行无源复制的菌株对古生菌重组酶RADA的需求来进行测试。基因组技术将被用来确定R-环(Drop-seq)和D-环(使用标签RADA的芯片序列)的位置，在有和没有起源的菌株中。过度的R-环导致细菌和真核生物的基因组不稳定，因为转录-复制冲突。我们将改造一种Haloferax Volcanii菌株，使其逆转一个高转录的rrn rRNA操纵子的方向，使RNA聚合酶与来自附近来源的DNA复制叉发生碰撞。或者，收敛的RRN操纵子将被用来研究转录-转录冲突。由聚合酶碰撞引起的基因组重排将通过单分子长读纳米孔测序来检测。最后，我们将研究RNA聚合酶回溯在R环形成中的作用。在细菌中，从R-环启动DNA复制需要从模板中移除停滞的RNA聚合酶的转录因子。我们将通过检测古生菌RNA聚合酶抑制剂在R环形成中的作用来测试Haloferax Volcanus II是否属于这种情况。

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.