Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par⁻). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par⁻ and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB.

中文翻译：

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PriA和PriC中残基的突变分析影响其与大肠杆菌K-12中SSB相互作用的能力。

大肠杆菌PriA和PriC识别废弃的复制叉，并将DnaB复制解旋酶直接重载到涂有单链DNA结合蛋白（SSB）的落后链模板上。生物化学和结构研究表明PriA和PriC均与SSB的C末端发生物理相互作用。在体外，这些相互作用触发了ssDNA上SSB的重塑。priA341（R697A）和priC351（R155A）在体外否定了SSB重塑反应。质粒携带的priC351（R155A）不补充priC303 :: kan和priA341（R697A）尚未经过互补性测试。在这里，我们通过将priA341（R697A），priA344（R697E），priA345（Q701E）和priC351（R155A）置于染色体上并鉴定突变菌株，进一步研究了PriA和PriC的SSB结合口袋。所有三个priA突变体的行为均类似于野生型。在一Δ priB株，突变引起在SOS表达，细胞大小适度增加，和缺陷类核分区（帕^- ）。SSB的过量生产部分抑制了priA341（R697A）和priA344（R697E）的这些表型。该priC351（R155A）突变体的行为符合预期：单个突变体中没有表型，并且当此突变体与ΔpriB结合使用时，存在严重的生长缺陷。该分析priBC突变揭示了两个细胞群：一类与野生型的表型和那些有非常丝状和帕^-和具有高SOS表达。我们的结论是在体内，priC351（R155A）来识别用于PRIC必需残基和功能，即PRIA R697和Q701仅在不存在PriB的是重要的，该蛋白质的该区域可具有与SSB一个复杂的关系。