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

该项目将研究R环在Haloferax volcanii无起点DNA复制中的作用，以及这种替代DNA复制模式对基因组稳定性的影响。DNA复制起始于称为起点的染色体序列。复制起源被认为是必不可少的，但我们已经表明，在Haloferax volcano ii中，一个成员的生物，没有起源的生命是可能的。DNA复制的机制在古生菌和真核生物中惊人地相似。从Haloferax volcanii的主染色体上删除所有复制起点不仅是可能的，而且会导致加速生长。我们以前已经表明，在没有起源的情况下，DNA复制的启动涉及同源重组，因为它依赖于古细菌重组酶RadA。同源重组涉及一个三链DNA结构的形成，称为D-环，这是由RadA催化的。我们现在已经获得了新的数据，显示了转录在复制中的作用，没有起点。沃氏嗜盐菌的基因组由一条具有三个起源的染色体和三条各具有一个起源的微型染色体组成。染色体上的所有起源都可以删除，但微型染色体pHV 3上的起源不能删除。值得注意的是，pHV 3上的转录水平非常低。我们已经在pHV 3上设计了一个高活性的启动子来增加转录，并且发现现在可以删除该起始点。这表明R环的作用。R环是由DNA：RNA杂合体组成的三链结构，它们通常是由于转录失败而形成的。R环具有直接从RNA链的3' OH端引发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有关，而DNA断裂又可以通过同源重组来引发DNA复制。最初的目的是确定R环是直接引发无起点的DNA复制，还是通过同源重组间接起作用。这将通过确定依赖于转录进行无起点复制的菌株中对古细菌重组酶RadA的需求来进行测试。基因组技术将用于确定R环（DRIP-seq）和D环（ChIP-seq使用标记的RadA）的位点，在有和没有起源的菌株中。由于转录-复制冲突，过多的R环导致细菌和真核生物的基因组不稳定。我们将设计一种沃氏嗜盐菌菌株，使高度转录的rrn rRNA操纵子转向，这样RNA聚合酶就能与附近起源的DNA复制叉发生碰撞。或者，会聚rrn操纵子将用于研究转录-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段纳米孔测序来检测。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从R环开始DNA复制所必需的。我们将测试这是否是Haloferax volcano ii的情况下，通过研究古细菌RNA聚合酶抑制剂在R环形成的作用。

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.