Oxidative DNA damage stalls the human mitochondrial replisome

• Published in: Scientific reports Vol. 6; no. 1; p. 28942
• Main Authors: Stojkovič, Gorazd, Makarova, Alena V., Wanrooij, Paulina H., Forslund, Josefin, Burgers, Peter M., Wanrooij, Sjoerd
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.07.2016; Nature Publishing Group
• Subjects: 631/45/147; 631/45/607/1159; 82/29; DNA Damage; DNA Helicases - metabolism; DNA polymerase; DNA Polymerase gamma - metabolism; DNA Primase - metabolism; DNA Replication; DNA, Mitochondrial - genetics; DNA-Binding Proteins - metabolism; DNA-Directed DNA Polymerase - metabolism; Humanities and Social Sciences; Humans; Mitochondria; Mitochondria - enzymology; Mitochondria - genetics; Mitochondrial DNA; Mitochondrial Proteins - metabolism; multidisciplinary; Multifunctional Enzymes - metabolism; Oxidative Stress; RNA polymerase; Science
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep28942

Oxidative stress is capable of causing damage to various cellular constituents, including DNA. There is however limited knowledge on how oxidative stress influences mitochondrial DNA and its replication. Here, we have used purified mtDNA replication proteins, i.e . DNA polymerase γ holoenzyme, the mitochondrial single-stranded DNA binding protein mtSSB, the replicative helicase Twinkle and the proposed mitochondrial translesion synthesis polymerase PrimPol to study lesion bypass synthesis on oxidative damage-containing DNA templates. Our studies were carried out at dNTP levels representative of those prevailing either in cycling or in non-dividing cells. At dNTP concentrations that mimic those in cycling cells, the replication machinery showed substantial stalling at sites of damage and these problems were further exacerbated at the lower dNTP concentrations present in resting cells. PrimPol, the translesion synthesis polymerase identified inside mammalian mitochondria, did not promote mtDNA replication fork bypass of the damage. This argues against a conventional role for PrimPol as a mitochondrial translesion synthesis DNA polymerase for oxidative DNA damage; however, we show that Twinkle, the mtDNA replicative helicase, is able to stimulate PrimPol DNA synthesis in vitro , suggestive of an as yet unidentified role of PrimPol in mtDNA metabolism.