National Centre for Biological Sciences, Tata Institute of Fundamental Research, Gandhi Krishi Vigyan Kendra, Bengaluru, Karnataka, India.
School of Life Science, The University of Trans-Disciplinary Health Sciences & Technology (TDU), Bengaluru, Karnataka, India.
mSphere. 2020 Feb 26;5(1):e00939-19. doi: 10.1128/mSphere.00939-19.
The bacterium can initiate replication in the absence of the replication initiator protein DnaA and/or the canonical origin of replication in a background. This phenomenon, which can be primed by R-loops, is called constitutive stable DNA replication (cSDR). Whether DNA replication during cSDR initiates in a stochastic manner through the length of the chromosome or at specific sites and how can find adaptations to loss of fitness caused by cSDR remain inadequately answered. We use laboratory evolution experiments of strains followed by deep sequencing to show that DNA replication preferentially initiates within a broad region located ∼0.4 to 0.7 Mb clockwise of This region includes many bisulfite-sensitive sites, which have been previously defined as R-loop-forming regions, and includes a site containing sequence motifs that favor R-loop formation. Initiation from this region would result in head-on replication-transcription conflicts at rRNA loci. Inversions of these rRNA loci, which can partly resolve these conflicts, help the bacterium suppress the fitness defects of cSDR. These inversions partially restore the gene expression changes brought about by cSDR. The inversion, however, increases the possibility of conflicts at essential mRNA genes, which would utilize only a minuscule fraction of RNA polymerase molecules, most of which transcribe rRNA genes. Whether subsequent adaptive strategies would attempt to resolve these conflicts remains an open question. The bacterium can replicate its DNA even in the absence of the molecules that are required for canonical replication initiation. This often requires the formation of RNA-DNA hybrid structures and is referred to as constitutive stable DNA replication (cSDR). Where on the chromosome does cSDR initiate? We answer this question using laboratory evolution experiments and genomics and show that selection favors cSDR initiation predominantly at a region ∼0.6 Mb clockwise of Initiation from this site will result in more head-on collisions of DNA polymerase with RNA polymerase operating on rRNA loci. The bacterium adapts to this problem by inverting a region of the genome including several rRNA loci such that head-on collisions between the two polymerases are minimized. Understanding such evolutionary strategies in the context of cSDR can provide insights into the potential causes of resistance to antibiotics that target initiation of DNA replication.
这种细菌可以在没有复制起始蛋白 DnaA 和/或经典复制起点的情况下启动复制，并且在背景下可以被 R 环引发。这种现象被称为组成型稳定 DNA 复制（cSDR）。cSDR 期间的 DNA 复制是通过染色体的长度随机起始，还是在特定位置起始，以及如何适应 cSDR 引起的适应性降低，仍然没有得到充分回答。我们使用实验室进化实验和深度测序来展示，DNA 复制优先在一个宽区域起始，该区域位于 顺时针方向约 0.4 到 0.7 Mb 处。该区域包含许多亚硫酸氢盐敏感位点，这些位点以前被定义为 R 环形成区域，并且包含一个包含有利于 R 环形成的序列基序的位点。从该区域起始会导致 rRNA 基因座处的复制-转录冲突。这些 rRNA 基因座的倒位可以部分解决这些冲突，从而帮助细菌抑制 cSDR 的适应性降低缺陷。这些倒位部分恢复了 cSDR 带来的基因表达变化。然而，倒位增加了必需 mRNA 基因处冲突的可能性，这些基因只利用 RNA 聚合酶分子的极小部分，而大部分 RNA 聚合酶转录 rRNA 基因。随后的适应性策略是否会试图解决这些冲突仍然是一个悬而未决的问题。细菌即使在没有用于经典复制起始的分子的情况下也可以复制其 DNA。这通常需要形成 RNA-DNA 杂交结构，并且被称为组成型稳定 DNA 复制（cSDR）。cSDR 在哪里起始？我们使用实验室进化实验和基因组学回答了这个问题，并表明选择有利于 cSDR 主要在 顺时针方向约 0.6 Mb 处的一个区域起始。从该位点起始将导致 DNA 聚合酶与在 rRNA 基因座上操作的 RNA 聚合酶之间更直接的碰撞。细菌通过倒位包括几个 rRNA 基因座在内的基因组区域来适应这个问题，从而最小化两个聚合酶之间的直接碰撞。在 cSDR 的背景下理解这种进化策略可以为理解抗生素耐药性的潜在原因提供线索，这些抗生素的靶标是 DNA 复制的起始。
