Figure 5: Failure to ligate can lead to nicks and linear mtDNA deletions.

(a) MtDNA was analysed on a 0.4% agarose gel in the absence (left panel) or the presence (right panel) of EtBr. The presence of EtBr in the agarose gel facilitates differentiation between nicked and closed circular dsDNA molecules. The mtDNA was detected by Southern blotting using probes against the major or minor arc. Results were confirmed three times with two independent batches of genomic DNA preparations. (b) A schematic representation of the PCR template including the D-loop region. Arrows and black bars show the PCR amplification regions. (c) The relative ratio of DNA copies of H-/L-strands in WT and mutator (EXO-) mice. Asterisks represent significant differences compared with WT (n=3; *P≤0.05, Student’s t-test). (d) Model of mtDNA linear deletion formation. Failure to ligate at OriH leads to a nick in the H-strand (left panel). The nick does not inhibit initiation of a new round of replication from OriH, as the intact L-strand is used as template for H-strand synthesis. DNA synthesis initiated at OriH may therefore continue full circle and create a new DNA molecule with a nick at OriH (right upper panel). In contrast, DNA synthesis initiated at OriL will result in linear, deleted fragments. In this later case, L-strand DNA synthesis will initiate from OriL and use the H-strand as template. DNA synthesis will reach the nick near OriH and a double-strand break will be formed. The molecule formed will also contain an unstable ssDNA region that will be degraded, leaving a linear double-stranded product spanning the major arc (right lower panel). Failure to ligate at OriH will thus result in the same round of replication generating two different replication products; a circular, nicked mtDNA and a linear, deleted fragment spanning OriH and OriL. Solid back line, H-strand; dashed black line, nascent H-strand; solid red line, L-strand; dashed red line, nascent L-strand.