Fig. 5. Analysis of early mp1 replication by an in vitro replication assay. (A) 2-DE of a construct harbouring the dso1 origin sequence, which was incubated in vitro for 60 min with a mitochondrial enzyme complex in the presence or absence of 10 µM NTPs. (B) Time course of mp1 in vitro replication using the dso1 construct and vector as a control. The results are the means of three experiments. (C) EM analysis of in vitro replication intermediates revealed R-loops (arrow) after 10 min. (D) Typical RCs were observed after 30 min. Bar = 0.5 kb. (E) Hypothetical model for the initiation of mp1 RCR involving an R-loop transcribed from ORF2 (see text).