Replication Pauses of the Wild-Type and Mutant Mitochondrial DNA Polymerase Gamma: A Simulation Study

The activity of polymerase γ is complicated, involving both correct and incorrect DNA polymerization events, exonuclease activity, and the disassociation of the polymerase:DNA complex. Pausing of pol-γ might increase the chance of deletion and depletion of mitochondrial DNA. We have developed a stochastic simulation of pol-γ that models its activities on the level of individual nucleotides for the replication of mtDNA. This method gives us insights into the pausing of two pol-γ variants: the A467T substitution that causes PEO and Alpers syndrome, and the exonuclease deficient pol-γ (exo−) in premature aging mouse models. To measure the pausing, we analyzed simulation results for the longest time for the polymerase to move forward one nucleotide along the DNA strand. Our model of the exo− polymerase had extremely long pauses, with a 30 to 300-fold increase in the time required for the longest single forward step compared to the wild-type, while the naturally occurring A467T variant showed at most a doubling in the length of the pauses compared to the wild-type. We identified the cause of these differences in the polymerase pausing time to be the number of disassociations occurring in each forward step of the polymerase. Author Summary: As a polymerase copies a DNA strand, it may briefly pause. The pauses of the polymerase are important factors in the formation of large-scale deletions in mitochondrial DNA, a major cause of pathogenicity in mitochondrial diseases. To investigate these pauses, we have developed a simulation of the function of the mitochondrial DNA polymerase. As well as the wild-type polymerase, we consider two important mutant forms of the polymerase. Based on the simulation we predict that one of these two mutants would experience severe polymerase pauses, while the second would have only slightly longer pauses than the wild-type. This is important information for understanding the differences in pathogenicity between these two mutant polymerases. Furthermore, based on the simulation we determine that a particular reaction of the polymerase, the disassociation of the polymerase:DNA complex, is the most critical reaction determining the length of the polymerase pauses. Of all of the complex functions of the polymerase, the disassociation reaction has had the least attention experimentally and medically. Our simulation model points out that this neglected behavior of the polymerase should be examined more carefully in future research.