title: MGME1 processes flaps into ligatable nicks in concert with DNA polymerase γ during mtDNA replication.
abstract: Recently, MGME1 was identified as a mitochondrial DNA nuclease with preference for single-stranded DNA (ssDNA) substrates. Loss-of-function mutations in patients lead to mitochondrial disease with DNA depletion, deletions, duplications and rearrangements. Here, we assess the biochemical role of MGME1 in the processing of flap intermediates during mitochondrial DNA replication using reconstituted systems. We show that MGME1 can cleave flaps to enable efficient ligation of newly replicated DNA strands in combination with POLγ. MGME1 generates a pool of imprecisely cut products (short flaps, nicks and gaps) that are converted to ligatable nicks by POLγ through extension or excision of the 3'-end strand. This is dependent on the 3'-5' exonuclease activity of POLγ which limits strand displacement activity and enables POLγ to back up to the nick by 3'-5' degradation. We also demonstrate that POLγ-driven strand displacement is sufficient to generate DNA- but not RNA-flap substrates suitable for MGME1 cleavage and ligation during replication. Our findings have implications for RNA primer removal models, the 5'-end processing of nascent DNA at OriH, and DNA repair.
authors.affiliation: Institute of Biomedicine, University of Gothenburg, P.O. Box 440, SE-405 30 Gothenburg, Sweden jennifer.uhler@medkem.gu.se.
authors.forename: Jay P
authors.initials: JP
authors.lastname: Uhler
authors.affiliation: Institute of Biomedicine, University of Gothenburg, P.O. Box 440, SE-405 30 Gothenburg, Sweden.
authors.forename: Christian
authors.initials: C
authors.lastname: Thörn
authors.affiliation: Institute of Biomedicine, University of Gothenburg, P.O. Box 440, SE-405 30 Gothenburg, Sweden.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Nicholls
authors.affiliation: Max Planck Institute for Biology of Ageing, 50391 Cologne, Germany.
authors.forename: Stanka
authors.initials: S
authors.lastname: Matic
authors.affiliation: Max Planck Institute for Biology of Ageing, 50391 Cologne, Germany.
authors.forename: Dusanka
authors.initials: D
authors.lastname: Milenkovic
authors.affiliation: Institute of Biomedicine, University of Gothenburg, P.O. Box 440, SE-405 30 Gothenburg, Sweden.
authors.forename: Claes M
authors.initials: CM
authors.lastname: Gustafsson
authors.affiliation: Institute of Biomedicine, University of Gothenburg, P.O. Box 440, SE-405 30 Gothenburg, Sweden maria.falkenberg@medkem.gu.se.
authors.forename: Maria
authors.initials: M
authors.lastname: Falkenberg
mesh_terms: D002474:Cell-Free System / Q000378:metabolism; D003001:Cloning, Molecular; D053837:DNA Cleavage; D000074002:DNA Polymerase gamma; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D011994:Recombinant Proteins; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C579164:MGME1 protein, human