Rolling Circle Replication and Bypass of Damaged Nucleotides.

Faithful mitochondrial DNA (mtDNA) replication is critical for the proper function of the oxidative phosphorylation system. Problems with mtDNA maintenance, such as replication stalling upon encountering DNA damage, impair this vital function and can potentially lead to disease. An in vitro reconstituted mtDNA replication system can be used to investigate how the mtDNA replisome deals with, for example, oxidatively or UV-damaged DNA.

Known Unknowns of Mammalian Mitochondrial DNA Maintenance.

Mammalian mitochondrial DNA (mtDNA) replication and repair have been studied intensively for the last 50 years. Although recently advances in elucidating the molecular mechanisms of mtDNA maintenance and the proteins involved in these have been made, there are disturbing gaps between the existing theoretical models and experimental observations. Conflicting data and hypotheses exist about the role of RNA and ribonucleotides in mtDNA replication, but also about the priming of replication and the formation of pathological rearrangements.

Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired.

Incorporation of ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove incorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs.

PrimPol is required for replication reinitiation after mtDNA damage.

Eukaryotic PrimPol is a recently discovered DNA-dependent DNA primase and translesion synthesis DNA polymerase found in the nucleus and mitochondria. Although PrimPol has been shown to be required for repriming of stalled replication forks in the nucleus, its role in mitochondria has remained unresolved. Here we demonstrate in vivo and in vitro that PrimPol can reinitiate stalled mtDNA replication and can prime mtDNA replication from nonconventional origins.