Towards Efficient Reconstitution of the Human Mitochondrial DNA Replication Complex

Abstract

The minimal human mitochondrial DNA replisome, comprising mitochondrial DNA polymerase (Pol γ), mitochondrial DNA helicase (mtDNA helicase) and mitochondrial single-stranded binding protein (mtSSB) was reconstituted. Assembly of these replication factors on the minicircle template, resembling the replication fork in vivo, supported the rolling-circle DNA replication. Quantitative analysis of the product formed from the minicircle template with the radioactive label on the primer strand or on the incoming ribonucleotides yielded consistent results, which demonstrated the poor efficiency (less than 1% active fraction) of the reconstituted replisome described (Korhonen et al., 2003). We sought to screen conditions to optimize the assembly of the replisome. The optimized assembly of replisome improves the replication efficiency to ∼14%. The relative low efficiency may indicate a poor assembly of mtDNA helicase or that the mitochondrial replisome in vivo comprises unidentified essential components. Notably, we found that stimulation of processive DNA synthesis by mtSSB occurs over a narrow range of salt concentration. We obtained more quantitative reconstitution the human mitochondrial replisome using a small synthetic replication fork and measured the kinetics of coupled DNA unwinding and polymerization. Optimal reconstitution was achieved by preincubation of the DNA with the polymerase, helicase and two of the four deoxyribonucleotides. Following the addition of the remaining two nucleotides, 80% of the primers were extended with a half-life of 84 seconds.