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Rolling-circle replication is initiated by the Rep protein, which is encoded by the plasmid. It binds to the double-strand origin (DSO) of the plasmid. The protein then nicks one of the strands and remains covalently bound to the 5' phosphate end of the nicked strand. The free 3' hydroxyl end is released to serve as a primer for DNA synthesis by DNA polymerase III. Using the unnicked strand as a template, replication proceeds around the plasmid, displacing the nicked strand as single-stranded DNA. Once this single-stranded circle is complete, the 5' end which is attached to Rep and the displaced 3' end are ligated in a phosphotransferase reaction. The double-stranded circle is closed as well when the DNA Polymerase returns to the DSO. This probably involves creating a second nick and the host DNA ligase.
The replication of the displaced single strand starts at the single-strand origin (SSO), which is only synthesized when the displacement is almost complete. At the SSO, the RNA polymerase makes a primer for DNA polymerase III, which synthesizes the complementary strand. Once it is finished, DNA polymerase I replaces the RNA primer and DNA ligase joins the ends.
Date
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Author
Tobias Vornholt
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