Figure 6.

PFV intasome activity following preincubation in the presence of PCA. PFV intasomes with Cy5 labeled viral donor DNA were incubated for variable time at 37 °C with PCA but without target DNA. Following the preincubation, supercoiled plasmid target DNA was added to the reaction and incubated for a further 5 min. (a) Reaction products were separated by agarose gel electrophoresis stained with ethidium bromide (top panels) and imaged for Cy5 fluorescence (bottom panels). (b) Cy5 fluorescent products were analyzed and expressed as the fraction of fluorescent signal in each lane. (c) Quantitation of the rate of increase in Cy5 donor with and without PCA (rate of instability). The addition of PCA correlates with a decrease in the rate of donor accumulation. Reactions were performed in the presence of 5 mM PCA. Experiments were performed at least three times with at least two independent intasome preparations. Error bars indicate standard deviation. NC, nicked circle. L-CI, linear CI products. SC, supercoiled plasmid. S-CI, smear CI products. Donor, viral donor.