Leroux Maxime, Jani Niketa, Sandler Steven J
Department of Microbiology, University of Massachusetts at Amherst, Amherst, Massachusetts, USA.
Department of Microbiology, University of Massachusetts at Amherst, Amherst, Massachusetts, USA
J Bacteriol. 2017 Aug 8;199(17). doi: 10.1128/JB.00267-17. Print 2017 Sep 1.
The ability to restart broken DNA replication forks is essential across all domains of life. In , the , , , and genes encode the replication restart proteins (RRPs) to accomplish this task. PriA plays a critical role in replication restart such that its absence reveals a dramatic phenotype: poor growth, high basal levels of SOS expression, poorly partitioned nucleoids (Par), UV sensitivity, and recombination deficiency (Rec). PriA has 733 amino acids, and its structure is composed of six domains that enable it to bind to DNA replication fork-like structures, remodel the strands of DNA, interact with SSB (single-stranded DNA binding protein), PriB, and DnaT, and display ATPase, helicase, and translocase activities. We have characterized a new mutation called :: It is a composite mutation involving an insertion that truncates the protein within the winged-helix domain (at the 154th codon) and an ACG (Thr)-to-ATG (Met) mutation that allows reinitiation of translation at the 157th codon such that PriA is expressed in two pieces. :: phenotypes are like those of the wild type for growth, recombination, and UV resistance, revealing only a slightly increased level of SOS expression and defects in nucleoid partitioning in the mutant. Both parts of PriA are required for activity, and the N-terminal fragment can be optimized to yield wild-type activity. A deletion of the protease suppresses :: phenotypes. We hypothesize the two parts of PriA form a complex that supplies most of the PriA activity needed in the cell. PriA is a highly conserved multifunctional protein that plays a crucial role in the essential process of replication restart. Here we characterize an insertion mutation of with an intragenic suppressor such that it is now made in two parts. These two pieces split the winged-helix domain to separate the N-terminal 3' DNA-binding domain from the C-terminal domain of PriA. It is hypothesized that the two pieces form a complex that is capable of almost wild type function. The composite mutation leads to a moderate level of SOS expression and defects in partitioning of the chromosomes. Full function is restored by deletion of , suggesting that stability of this complex may be a reason for the partial phenotypes seen.
重新启动断裂的DNA复制叉的能力在生命的所有领域都是必不可少的。在[具体生物名称]中，[具体基因1]、[具体基因2]、[具体基因3]和[具体基因4]基因编码复制重新启动蛋白（RRP）来完成这项任务。PriA在复制重新启动中起关键作用，因此其缺失会揭示出一种显著的表型：生长不良、SOS表达的基础水平高、类核（Par）分配不良、紫外线敏感性和重组缺陷（Rec）。PriA有733个氨基酸，其结构由六个结构域组成，这些结构域使其能够结合到DNA复制叉样结构、重塑DNA链、与单链DNA结合蛋白（SSB）、PriB和DnaT相互作用，并表现出ATP酶、解旋酶和转位酶活性。我们鉴定了一个名为[具体名称]的新突变::它是一个复合突变，涉及一个插入，该插入在翼状螺旋结构域内（第154个密码子处）截断蛋白质，以及一个从ACG（苏氨酸）到ATG（甲硫氨酸）的突变，该突变允许在第157个密码子处重新启动翻译，使得PriA以两个片段的形式表达。::的表型在生长、重组和紫外线抗性方面与野生型相似，仅显示突变体中SOS表达水平略有增加和类核分配缺陷。PriA的两个部分都需要活性，并且N端片段可以优化以产生野生型活性。[具体蛋白酶名称]的缺失抑制了::的表型。我们假设PriA的两个部分形成一个复合物，该复合物提供细胞中所需的大部分PriA活性。PriA是一种高度保守的多功能蛋白质，在复制重新启动的基本过程中起关键作用。在这里，我们鉴定了一个带有基因内抑制子的[具体基因名称]插入突变，使得它现在以两个部分的形式产生。这两个片段将翼状螺旋结构域分开，将N端3' DNA结合结构域与PriA的C端结构域分离。据推测，这两个片段形成一个能够几乎发挥野生型[具体功能名称]功能的复合物。复合突变导致SOS表达处于中等水平以及染色体分配缺陷。通过缺失[具体基因名称]恢复了完全功能，这表明该复合物的稳定性可能是观察到部分表型的一个原因。
