A stem–loop structure, sequence non-specific, at the origin of DNA replication of porcine circovirus is essential for termination but not for initiation of rolling-circle DNA replication

A stem–loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. In this work, a head-to-tail tandem construct of porcine circovirus capable of generating unit-length genomic DNA in Escherichia coli was employed to examine the role of the stem–loop structure with respect to the RCR initiation and termination process. The advantage of using a head-to-tail tandem construct is that the initiation and termination sites for generation of the unit-length viral genomes are physically separated, which allows independent examination of the initiation/termination processes. Nucleotide substitution mutational analysis showed that a pair of inverted repeats capable of forming a stem–loop structure was essential for termination, but not for initiation. The results also demonstrated that it is the stem–loop configuration, not nucleotide sequence specificity, that is critical for terminating RCR DNA replication.