FIG. 5.
Effect of PRI1 RNAi on minicircle and maxicircle DNAs. (A) Kinetics of kDNA loss as determined by Southern blotting of total minicircles and maxicircles. After digestion with HindIII and XbaI, total cellular DNA (1 × 106 cell equivalents per lane) was fractionated on a 1.5% agarose gel containing 1 μg/ml ethidium bromide. Southern blots were probed for minicircle DNA, maxicircle fragment (Maxi), and a hexose transporter fragment as a loading control (Load). A 1.0-kb minicircle fragment (Mini) represents the full-length minicircle. (B) Phosphorimager quantitation of the relative levels of maxicircle and minicircle DNAs shown in panel A. (C) Effect of PRI1 RNAi on free minicircle replication intermediates. Total DNA was purified after various times of RNAi and then fractionated (5 × 105 cell equivalents per lane) on an agarose gel. Minicircle DNA was detected by Southern blotting using a minicircle probe. Intact kDNA does not enter the gel and remains in the well. The band near the top of the gel is due to chromosomal DNA. Covalently closed (CC), nicked/gapped (N/G), and catenated minicircles (cm) are indicated. (D) Phosphorimager quantitation of the relative levels of free minicircle species after various times of RNAi.