Hyperactive initiation of chromosomal replication in vivo and in vitro by a mutant initiator protein, DnaAcos, of Escherichia coli.
Hyperactive initiation of chromosomal replication in vivo and in vitro by a mutant initiator protein, DnaAcos, of Escherichia coli.
复制标题
大肠杆菌的突变起始蛋白 DnaAcos 在体内和体外过度活跃地启动染色体复制。
DOI：
10.1016/s0021-9258(18)99932-1
复制
发表时间：
1994
期刊：
影响因子：
--
通讯作者：
A. Kornberg
中科院分区：
文献类型：
--
作者：
T. Katayama;A. Kornberg
dna蛋白在大肠杆菌中启动基因组复制。一种冷敏感的dnaAcos突变体在限制性温度下引起过度起始而不增加DnaA蛋白水平。染色体起源（oriC）是dnaAcos产物致死性的关键。增加的起始活性被质粒（pBR322）上的多个拷贝oriC所中和。OriC质粒在dnaAcos突变体粗提取物中得到了高效的体外复制，其对dnaAcos蛋白的特异性活性比野生型提取物中的DnaA+蛋白高8倍。与dnaA+提取物中的复制一样，利福平和回转酶抑制剂抑制了dnaAcos提取物中oric依赖性复制。作为对照，不需要DnaA蛋白的单链噬菌体phi X174 DNA在DnaA +和dnaAcos细胞提取物中复制相似。因此，DnaAcos蛋白在oriC上的起始似乎在体内和体外都是高度激活的。
DnaA protein initiates genomic replication in Escherichia coli. A cold-sensitive dnaAcos mutant caused excessive initiation at a restrictive temperature without an increase in the level of DnaA protein. The chromosomal origin (oriC) was essential for the lethality caused by the dnaAcos product. Increased initiation activity was neutralized by multiple copies of oriC on a plasmid (pBR322). OriC plasmids were replicated efficiently in vitro in a crude extract prepared from a dnaAcos mutant, with a specific activity for the DnaAcos protein 8-fold greater than that for the DnaA+ protein in a wild-type extract. OriC-dependent replication in the dnaAcos extract was inhibited by rifampicin and by gyrase inhibitors as was replication in the dnaA+ extract. As a control, replication of single-stranded phage phi X174 DNA, which did not require DnaA protein, was similar in extracts prepared from dnaA+ and dnaAcos cells. Thus, initiation at oriC by DnaAcos protein appears to be highly activated both in vivo and in vitro.