Open AccessInitiation of unidirectional CoIE2 DNA replication by a unique priming mechanism
Author(s) -
Seiji Takechi,
Tateo Itoh
Publication year - 1995
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/23.20.4196
Subject(s) - dnag , primase , biology , dna replication , dna clamp , primer (cosmetics) , dna polymerase , dna polymerase ii , dna , coding strand , okazaki fragments , replisome , prokaryotic dna replication , eukaryotic dna replication , sense strand , polymerase , circular bacterial chromosome , control of chromosome duplication , dna polymerase i , genetics , rna , gene , reverse transcriptase , chemistry , organic chemistry
The ColE2 DNA can be replicated in an in vitro system consisting of a crude extract of Escherichia coli cells. DNA synthesis requires a plasmid-coded protein (Rep) and host DNA polymerase I but not host RNA polymerase. Replication starts at a fixed region containing the origin and proceeds unidirectionally. The leading- and lagging-strand DNA fragments synthesized around the origin were identified from early replicative intermediates. The 5' end of the leading-strand DNA fragment was mapped at a unique position in the minimal origin and carried RNA of a few residues. The results suggested that the initiation of the leading-strand DNA synthesis does not require the host DnaG protein. Thus the Rep protein itself seems to be a primase. Synthesis of the primer RNA at a fixed site in the origin region on a double-stranded DNA template is a unique property of the ColE2 Rep protein among other known primases. The 3' end of the lagging-strand DNA fragment was mapped at a unique position just at the end of the minimal origin region. Termination of the lagging-strand DNA fragment at that position seems to be the mechanism of the unidirectional replication of ColE2 plasmid.