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

该项目将研究r环在火山盐藓无起源DNA复制中的作用，以及这种DNA复制的替代模式对基因组稳定性的影响。DNA复制开始于染色体序列的起点。复制起源被认为是必不可少的，但我们已经证明，在古生菌的成员火山盐藻中，没有起源的生命是可能的。除了真核生物和细菌外，古细菌是生命的第三个领域。古细菌和真核生物的DNA复制机制惊人地相似。火山盐铁鱼主染色体上所有复制起点的缺失不仅是可能的，而且会导致生长加速。我们之前已经证明，在没有起源的情况下，DNA复制的起始涉及同源重组，因为它依赖于古菌重组酶RadA。同源重组涉及一种称为d环的三链DNA结构的形成，这是由RadA催化的。我们现在获得了新的数据，显示了转录在无起点复制中的作用。火山盐铁鱼的基因组由一条染色体和三条小染色体组成，染色体各有一个起源。染色体上的所有起源都可以被删除，但迷你染色体pHV3上的起源不能被删除。值得注意的是，pHV3的转录水平非常低。我们在pHV3上设计了一个高活性启动子来增加转录，并发现现在可以删除起源。这表明了r环的作用。r环是由DNA和RNA杂交而成的三链结构，它们通常是由于转录失败而形成的。r -环具有从RNA链的3' OH端直接启动DNA复制的潜力。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断裂反过来又可以通过同源重组来启动DNA复制。最初的目的是确定r环是直接启动无起源DNA复制，还是通过同源重组间接起作用。这将通过确定依赖转录进行无起点复制的菌株对古菌重组酶RadA的需求来验证。基因组技术将用于确定r环（DRIP-seq）和d环（ChIP-seq，使用标记RadA）在有和没有起源的菌株中的位置。在细菌和真核生物中，由于转录-复制冲突，过量的r环导致基因组不稳定。我们将设计一种火山盐藻菌株，使其翻转高度转录的rrn rRNA操纵子的方向，从而使RNA聚合酶与附近起源的DNA复制叉发生碰撞。或者，收敛的rrn操作子将用于研究转录-转录冲突。聚合酶碰撞导致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，从模板中移除停滞的RNA聚合酶的转录因子是r环DNA复制起始所必需的。我们将通过检查古细菌RNA聚合酶抑制剂在r -环形成中的作用来测试火山盐铁ax是否是这种情况。

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.