title: Defects in maintenance of mitochondrial DNA are associated with intramitochondrial nucleotide imbalances.
abstract: Defects in mtDNA maintenance range from fatal multisystem childhood diseases, such as Alpers syndrome, to milder diseases in adults, including mtDNA depletion syndromes (MDS) and familial progressive external ophthalmoplegia (AdPEO). Most are associated with defects in genes involved in mitochondrial deoxynucleotide metabolism or utilization, such as mutations in thymidine kinase 2 (TK2) as well as the mtDNA replicative helicase, Twinkle and gamma polymerase (POLG). We have developed an in vitro system to measure incorporation of radiolabelled dNTPs into mitochondria of saponin permeabilized cells. We used this to compare the rates of mtDNA synthesis in cells from 12 patients with diseases of mtDNA maintenance. We observed reduced incorporation of exogenous alpha (32)P-dTTP in fibroblasts from a patient with Alpers syndrome associated with the A467T substitution in POLG, a patient with dGK mutations, and a patient with mtDNA depletion of unknown origin compared to controls. However, incorporation of alpha (32)P-dTTP relative to either cell doubling time or alpha (32)P-dCTP incorporation was increased in patients with thymidine kinase deficiency or PEO as the result of TWINKLE mutations compared with controls. The specific activity of newly synthesized mtDNA depends on the size of the endogenous pool diluting the exogenous labelled nucleotide. Our result is consistent with a deficiency in the intramitochondrial pool of dTTP relative to dCTP in cells from patients with TK2 deficiency and TWINKLE mutations. Such DNA precursor asymmetry could cause pausing of the replication complex and hence exacerbate the propensity for age-related mtDNA mutations. Because deviations from the normal concentrations of dNTPs are known to be mutagenic, we suggest that intramitochondrial nucleotide imbalance could underlie the multiple mtDNA mutations observed in these patients.
authors.affiliation: Mitochondrial Genetics Group, Nuffield Department of Obstetrics and Gynaecology, Level 3, Women's Centre,The John Radcliffe Hospital, Oxford OX3 9DU, UK.
authors.forename: Neil
authors.initials: N
authors.lastname: Ashley
authors.forename: Susan
authors.initials: S
authors.lastname: Adams
authors.forename: Abdelhamid
authors.initials: A
authors.lastname: Slama
authors.forename: Massimo
authors.initials: M
authors.lastname: Zeviani
authors.forename: Anu
authors.initials: A
authors.lastname: Suomalainen
authors.forename: Antonio L
authors.initials: AL
authors.lastname: Andreu
authors.forename: Robert K
authors.initials: RK
authors.lastname: Naviaux
authors.forename: Joanna
authors.initials: J
authors.lastname: Poulton
mesh_terms: D002463:Cell Membrane Permeability; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D000074002:DNA Polymerase gamma; D004272:DNA, Mitochondrial / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism*; D006801:Humans; D028361:Mitochondrial Diseases / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins; D008954:Models, Biological; D009154:Mutation; D010761:Phosphorus Radioisotopes; D013577:Syndrome; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism
chemical_list: D004272:DNA, Mitochondrial; D003845:Deoxycytosine Nucleotides; D003854:Deoxyribonucleotides; D024101:Mitochondrial Proteins; D010761:Phosphorus Radioisotopes; D013942:Thymine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; C105339:thymidine kinase 2; D013937:Thymidine Kinase; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C413027:POLG protein, human; D004265:DNA Helicases; C431576:TWNK protein, human; C024157:thymidine 5'-triphosphate