Initiation of unidirectional ColE2 DNA replication by a unique priming mechanism.

AUTOR(ES)

Takechi, S

RESUMO

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.

Documentos Relacionados

• Identification of a plasmid-coded protein required for initiation of ColE2 DNA replication.
• Primer RNA synthesis by plasmid-specified Rep protein for initiation of ColE2 DNA replication.
• Strand-Specific Supercoiled DNA-Protein Relaxation Complexes: Comparison of the Complexes of Bacterial Plasmids ColE1 and ColE2
• Control of ColE2 DNA replication: in vitro binding of the antisense RNA to the Rep mRNA.
• Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H.