Fig. 3.

mtDNA RIs are composed of two classes of molecules with different sensitivity to nucleases. (A) 2D-AGE analysis of O_H-containing fragments of mouse liver mtDNA digested with the restriction enzyme BclI and treated with nucleases as follows: left untreated (U) or treated with low levels of RNase H (↓RH), standard levels of RNase H (RH) or RH and S1 nuclease (S1) [Figure 5 in (45) for details]. These panels are reuse of those presented in Yasukawa et al. (45). Interpretations of arcs visualized using Southern hybridization are shown below each panel. Prominent bubble arcs (bubble) and SMY arcs (SMYs) were modified by RNase H and were degraded with the addition of S1 nuclease, suggesting that they are ribonucleotide-containing RIs. On the other hand, a fraction of the bubble arcs and Y arcs were resistant to nuclease treatments, indicating that such RIs have properties of those from conventional coupled leading- and lagging-strand DNA synthesis. Nuclease-resistant bubble arcs were also detected from non-NCR-containing fragments. 1N indicates non-replicating fragments. (B) Schematic drawings of proposed molecular structures of arcs observed in (A). ‘Orange arcs’ and ‘green and black arcs’ indicate arcs drawn in orange colour and arcs in green and black colours in (A), respectively.