title: Replication factors transiently associate with mtDNA at the mitochondrial inner membrane to facilitate replication.
abstract: Mitochondrial DNA (mtDNA) is organized in discrete protein-DNA complexes, nucleoids, that are usually considered to be mitochondrial-inner-membrane associated. Here we addressed the association of replication factors with nucleoids and show that endogenous mtDNA helicase Twinkle and single-stranded DNA-binding protein, mtSSB, co-localize only with a subset of nucleoids. Using nucleotide analogs to identify replicating mtDNA in situ, the fraction of label-positive nucleoids that is Twinkle/mtSSB positive, is highest with the shortest labeling-pulse. In addition, the recruitment of mtSSB is shown to be Twinkle dependent. These proteins thus transiently associate with mtDNA in an ordered manner to facilitate replication. To understand the nature of mtDNA replication complexes, we examined nucleoid protein membrane association and show that endogenous Twinkle is firmly membrane associated even in the absence of mtDNA, whereas mtSSB and other nucleoid-associated proteins are found in both membrane-bound and soluble fractions. Likewise, a substantial amount of mtDNA is found as soluble or loosely membrane bound. We show that, by manipulation of Twinkle levels, mtDNA membrane association is partially dependent on Twinkle. Our results thus show that Twinkle recruits or is assembled with mtDNA at the inner membrane to form a replication platform and amount to the first clear demonstration that nucleoids are dynamic both in composition and concurrent activity.
authors.affiliation: FinMIT Centre of Excellence, Institute of Biomedical Technology & Tampere University Hospital, Pirkanmaa Hospital District, FI-33014 University of Tampere, Finland and Department of Pediatrics, Institute for Genetic and Metabolic Disease, Radboud University Medical Centre, Geert Grooteplein 10, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands.
authors.forename: Nina
authors.initials: N
authors.lastname: Rajala
authors.forename: Joachim M
authors.initials: JM
authors.lastname: Gerhold
authors.forename: Peter
authors.initials: P
authors.lastname: Martinsson
authors.forename: Alexey
authors.initials: A
authors.lastname: Klymov
authors.forename: Johannes N
authors.initials: JN
authors.lastname: Spelbrink
mesh_terms: D002460:Cell Line; D002478:Cells, Cultured; D004265:DNA Helicases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000096:biosynthesis* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; C504091:NABP2 protein, human; D004265:DNA Helicases; C431576:TWNK protein, human