Sutton M D, Kaguni J M
Department of Biochemistry, Michigan State University, East Lansing 48824-1319, USA.
J Mol Biol. 1997 Dec 12;274(4):546-61. doi: 10.1006/jmbi.1997.1425.
The Escherichia coli DnaA protein is a sequence-specific DNA binding protein that promotes the initiation of replication of the bacterial chromosome, and of several plasmids including pSC101. Twenty-eight novel missense mutations of the E. coli dnaA gene were isolated by selecting for their inability to replicate a derivative of pSC101 when contained in a lambda vector. Characterization of these as well as seven novel nonsense mutations and one in-frame deletion mutation are described here. Results suggest that E. coli DnaA protein contains four functional domains. Mutations that affect residues in the P-loop or Walker A motif thought to be involved in ATP binding identify one domain. The second domain maps to a region near the C terminus and is involved in DNA binding. The function of the third domain that maps near the N terminus is unknown but may be involved in the ability of DnaA protein to oligomerize. Two alleles encoding different truncated gene products retained the ability to promote replication from the pSC101 origin but not oriC, identifying a fourth domain dispensable for replication of pSC101 but essential for replication from the bacterial chromosomal origin, oriC.
大肠杆菌DnaA蛋白是一种序列特异性DNA结合蛋白，可促进细菌染色体以及包括pSC101在内的几种质粒的复制起始。通过筛选当包含在λ载体中时无法复制pSC101衍生物的情况，分离出了28个大肠杆菌dnaA基因的新型错义突变。本文描述了这些突变以及7个新型无义突变和1个框内缺失突变的特征。结果表明，大肠杆菌DnaA蛋白包含四个功能域。影响P环或沃克A基序中被认为与ATP结合有关的残基的突变确定了一个功能域。第二个功能域定位在靠近C末端的区域，与DNA结合有关。定位在靠近N末端的第三个功能域的功能未知，但可能与DnaA蛋白寡聚化的能力有关。两个编码不同截短基因产物的等位基因保留了从pSC101起始位点促进复制的能力，但不能从oriC起始位点促进复制，这确定了第四个功能域，该功能域对于pSC101的复制是可有可无的，但对于从细菌染色体起始位点oriC进行复制是必不可少的。
