title: Oxidative DNA damage stalls the human mitochondrial replisome.
abstract: 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.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
authors.forename: Gorazd
authors.initials: G
authors.lastname: Stojkovič
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.|Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.
authors.forename: Alena V
authors.initials: AV
authors.lastname: Makarova
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.|Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Paulina H
authors.initials: PH
authors.lastname: Wanrooij
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
authors.forename: Josefin
authors.initials: J
authors.lastname: Forslund
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Burgers
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.|Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Sjoerd
authors.initials: S
authors.lastname: Wanrooij
mesh_terms: D004249:DNA Damage; D004265:DNA Helicases / Q000378:metabolism; D000074002:DNA Polymerase gamma / Q000378:metabolism; D019915:DNA Primase / Q000378:metabolism; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D064251:Multifunctional Enzymes / Q000378:metabolism; D018384:Oxidative Stress
chemical_list: D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; D064251:Multifunctional Enzymes; C504091:NABP2 protein, human; D019915:DNA Primase; C585901:PrimPol protein, human; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases; C431576:TWNK protein, human