当前位置: X-MOL 学术Mol. Microbiol. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Novel Escherichia coli active site dnaE alleles with altered base and sugar selectivity
Molecular Microbiology ( IF 3.6 ) Pub Date : 2021-06-28 , DOI: 10.1111/mmi.14779
Alexandra Vaisman 1 , Krystian Łazowski 2 , Martin A M Reijns 3 , Erin Walsh 1 , John P McDonald 1 , Kristiniana C Moreno 1 , Dominic R Quiros 1 , Marlen Schmidt 4 , Harald Kranz 4 , Wei Yang 5 , Karolina Makiela-Dzbenska 2 , Roger Woodgate 1
Affiliation  

The Escherichia coli dnaE gene encodes the α-catalytic subunit (pol IIIα) of DNA polymerase III, the cell’s main replicase. Like all high-fidelity DNA polymerases, pol III possesses stringent base and sugar discrimination. The latter is mediated by a so-called “steric gate” residue in the active site of the polymerase that physically clashes with the 2′-OH of an incoming ribonucleotide. Our structural modeling data suggest that H760 is the steric gate residue in E.coli pol IIIα. To understand how H760 and the adjacent S759 residue help maintain genome stability, we generated DNA fragments in which the codons for H760 or S759 were systematically changed to the other nineteen naturally occurring amino acids and attempted to clone them into a plasmid expressing pol III core (α-θ-ε subunits). Of the possible 38 mutants, only nine were successfully sub-cloned: three with substitutions at H760 and 6 with substitutions at S759. Three of the plasmid-encoded alleles, S759C, S759N, and S759T, exhibited mild to moderate mutator activity and were moved onto the chromosome for further characterization. These studies revealed altered phenotypes regarding deoxyribonucleotide base selectivity and ribonucleotide discrimination. We believe that these are the first dnaE mutants with such phenotypes to be reported in the literature.

中文翻译:

具有改变的碱基和糖选择性的新型大肠杆菌活性位点 dnaE 等位基因

大肠杆菌 dnaE基因编码 DNA 聚合酶 III的α 催化亚基 (pol IIIα),这是细胞的主要复制酶。与所有高保真 DNA 聚合酶一样,pol III 具有严格的碱基和糖区分能力。后者由聚合酶活性位点中的所谓“空间门”残基介导,该残基与进入的核糖核苷酸的 2'-OH 发生物理冲突。我们的结构建模数据表明 H760 是大肠杆菌中的空间门残基pol IIIα。为了了解 H760 和相邻的 S759 残基如何帮助维持基因组稳定性,我们生成了 DNA 片段,其中 H760 或 S759 的密码子系统地改变为其他 19 个天然存在的氨基酸,并试图将它们克隆到表达 pol III 核心的质粒中。 α-θ-ε 亚基)。在可能的 38 个突变体中,只有 9 个被成功亚克隆:3 个在 H760 处进行了替换,6 个在 S759 处进行了替换。质粒编码的三个等位基因 S759C、S759N 和 S759T 表现出轻度至中度的突变活性,并被移至染色体上以进行进一步表征。这些研究揭示了关于脱氧核糖核苷酸碱基选择性和核糖核苷酸辨别的改变的表型。我们相信这些是第一个dnaE具有这种表型的突变体将在文献中报道。
更新日期:2021-06-28
down
wechat
bug