Submitted to: Keystone Symposia
Publication Type: Abstract Only
Publication Acceptance Date: December 19, 2012
Publication Date: March 3, 2013
Citation: Cheung, A.K. 2013. Functions of Rep and Rep' during porcine circovirus rolling-circle replication [abstract]. DNA Replication and Recombination - training for proteins involved in eukaryotic DNA replication, Keystone Symposium, March 3-8, 2013, Banff, Canada. Paper No. Abs. x5 1017. p. 80. Technical Abstract: PCV replicates its single-stranded (ss) DNA genome via the rolling-circle replication (RCR) mechanism. In contrast to other RCR biological systems which utilize only one multi-functional protein (Rep) to replicate their respective genomes, PCV requires two proteins (Rep and Rep'). Rep and Rep' are identical at the N-terminal portion, but differ at the C-terminal portion. The fact that both proteins contain identical RCR motifs at the 5' portion but differ at the 3' portion, they are expected to exhibit some similar biological functions, but not all, under the same experimental conditions. In vitro, both Rep and Rep' exhibit nicking/joining activities in concert with the recognition sequence at the Ori. Each protein can covalently linked itself to the 5'-end of the nicked DNA after cleavage and the attached protein can re-ligate the nicked recognition sequence together in the presence of a stem-loop structure. However, the role(s) of each protein during PCV DNA replication is not known. Previously, it was demonstrated that unit-size PCV DNA can be excised from a head-to-tail (HTT) tandem genome construct inserted into a bacterial plasmid. The excision of ss-circular PCV genomes from the HTT construct is via the copy-release mechanism which requires the presence of two Oris and a functional Rep gene (with no involvement of Rep'). In this work, HTT constructs containing various combinations of functional Rep- and Rep'-genes were analyzed by 2D chloroquine gel/southern blots. The results showed that Rep is responsible for the excision of ss-circular genomes which are then converted to double-stranded (ds) open-circular (oc) molecules using the as yet unidentified minus-genome primers. Rep' plays a role in sealing the ds oc-molecules to become relaxed intermediates that are subsequently converted to covalently closed circular supercoiled DNAs by host enzymes and serve as templates for RCR to produce progeny PCV genomes.