Homologous recombination plays minor role in excision of unit-length viral genomes from head-to-tail direct tandem repeats of porcine circovirus during DNA replication in Escherichia coli.
In this report, we confirmed previous work that a theta-replicating bacterial plasmid containing 1.75 copies of genomic porcine circovirus (PCV) DNA in head-to-tail tandem (HTT) [a partial copy of PCV type 1 (PCV1), a complete copy of PCV type 2 (PCV2) and two origins of DNA replication (Ori)] yielded three different double-stranded DNA species when transformed into Escherichia coli: the input construct (U), the unit-length PCV1/PCV2 genome with a composite Ori lacking the plasmid vector (Q(RC)) and the remaining left-over 0.75 copy PCV1/PCV2 genome with a different composite Ori inserted in the plasmid vector (L(RC)). Replication of U was presumably via the theta-like replication mechanism utilizing the colicin E1 Ori, while derivation of L(RC) and Q(RC) was via the rolling-circle replication (RCR) copy-release mechanism and required the presence of two PCV Oris and the virus-encoded Rep protein. Essentially, excision of a unit-length PCV1/PCV2 genome (Q(RC)) via RCR from U yielded L(RC). Furthermore, we examined whether homologous recombination may also result in excision of a different type of unit-length PCV genome (Q(H)) from identical HTT constructs to generate L(H). Whereas the generation of L(RC) is Rep-protein-dependent, the generation of L(H) is Rep-protein-independent. Accordingly, the L(RC) and Q(RC) molecules derived from RCR possess different characteristics from the L(H) and Q(H) molecules generated via homologous recombination. In one of the studies in which both L(RC) and L(H) were generated simultaneously from the same HTT construct, out of 69 samples analyzed, 66 were derived via RCR and 3 were derived via homologous recombination. Thus, in comparison with RCR, homologous recombination plays a minor role in the excision of unit-length PCV genomes from HTT constructs in Escherichia coli.
Cheung
A K
AK
Virus and Prion Diseases of Livestock Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, Iowa 50010, USA. acheung@nadc.ars.usda.gov
D000818:Animals; D001483:Base Sequence; D018173:Circoviridae Infections; D017925:Circovirus / Q000235:genetics*; D053904:DNA Breaks, Single-Stranded; D004261:DNA Replication; D004926:Escherichia coli / Q000821:virology*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D010957:Plasmids; D011995:Recombination, Genetic; D018741:Replication Origin; D013552:Swine / Q000821:virology*; D020080:Tandem Repeat Sequences / Q000235:genetics*