Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii
Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii
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DOI：
10.1093/nar/27.22.4444
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发表时间：
1999-11-15
影响因子：
14.9
通讯作者：
Pisani, FM
中科院分区：
文献类型：
--
作者：
Desogus, G;Onesti, S;Pisani, FM
我们报告的鉴定和表征的DNA引物从嗜热产甲烷古菌詹氏甲烷球菌（Mjpri）。对该生物体的完整基因组序列的分析已经鉴定了编码与真核DNA引发酶的小亚基（聚合酶α-引发酶复合物的p50亚基）具有序列相似性的蛋白质的开放阅读框。该蛋白质已在大肠杆菌中过表达并纯化至接近同质。重组Mjpri能够在各种嘧啶单链DNA模板[poly（dT）和poly（dC）]上合成寡核糖核苷酸。该活性需要二价阳离子如Mg 2+、Mn 2+或Zn 2+，并且另外由单价阳离子K+刺激。多重序列比对显示，真核生物p50亚基中保守的大部分区域也存在于古细菌引发酶中，包括保守的带负电荷的残基，其已被证明是小鼠引发酶中催化所必需的。四个半胱氨酸残基，已假定构成一个假定的锌结合基序，两个是不存在于古细菌同源。这是第一份关于古细菌DNA引发酶的生化特性的报告。
We report the identification and characterisation of a DNA primase from the thermophilic methanogenic archaeon Methanococcus jannaschii (Mjpri). The analysis of the complete genome sequence of this organism has identified an open reading frame coding for a protein with sequence similarity to the small subunit of the eukaryotic DNA primase (the p50 subunit of the polymerase alpha-primase complex). This protein has been overexpressed in Escherichia coli and purified to near homogeneity. Recombinant Mjpri is able to synthesise oligoribonucleotides on various pyrimidine single-stranded DNA templates [poly(dT) and poly(dC)]. This activity requires divalent cations such Mg2+, Mn2+ or Zn2+, and is additionally stimulated by the monovalent cation K+. A multiple sequence alignment has revealed that most of the regions that are conserved in eukaryotic p50 subunits are also present in the archaeal primases, including the conserved negatively charged residues, which have been shown to be essential for catalysis in the mouse primase. Of the four cysteine residues that have been postulated to make up a putative Zn-binding motif, two are not present in the archaeal homologue. This is the first report on the biochemical characterisation of an archaeal DNA primase.