title: mtSSB may sequester UNG1 at mitochondrial ssDNA and delay uracil processing until the dsDNA conformation is restored.
abstract: Single-strand DNA binding proteins protect DNA from nucleolytic damage, prevent formation of secondary structures and prevent premature reannealing of DNA in DNA metabolic transactions. In eukaryotes, the nuclear single-strand DNA binding protein RPA is essential for chromosomal DNA replication and transcription and directly participates in several DNA repair processes by binding to and modulating the activity of repair factors. Much less is known about the involvement of the only mitochondrial single-strand binding protein mtSSB in the context of DNA repair. Here we demonstrate that mtSSB impedes excision of uracil and oxidative demethylation of 3meC in single-stranded DNA by UNG1 and ABH1, respectively, whereas excision by NEIL1 was partially inhibited. mtSSB also effectively inhibited nicking of single-stranded DNA by APE1 and ABH1 and partially inhibited the lyase activity of NEIL1. Finally we identified a putative surface motif in mtSSB that may recruit UNG1 to DNA-bound mtSSB. We suggest that the massive amount of mtSSB in mitochondria effectively prevents processing of uracil and other types of damaged bases to avoid introduction of nicks in single-stranded mtDNA formed during replication. Local enrichment of UNG1 at DNA-bound mtSSB may furthermore facilitate rapid access to- and processing of the damage once the dsDNA conformation is restored. This could be of potential biological importance, since mitochondria have no or limited capacity for homologous recombination to process nicks at the replication fork.
authors.affiliation: Department of Cancer Research and Molecular Medicine and the FUGE Proteomics Laboratory, Norwegian University of Science and Technology, Trondheim, Norway. kristee@stud.ntnu.no
authors.forename: Kristian
authors.initials: K
authors.lastname: Wollen Steen
authors.forename: Berit
authors.initials: B
authors.lastname: Doseth
authors.forename: Marianne
authors.initials: M
authors.lastname: P Westbye
authors.forename: Mansour
authors.initials: M
authors.lastname: Akbari
authors.forename: Dongchon
authors.initials: D
authors.lastname: Kang
authors.forename: Maria
authors.initials: M
authors.lastname: Falkenberg
authors.forename: Geir
authors.initials: G
authors.lastname: Slupphaug
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D002457:Cell Extracts; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D019175:DNA Methylation; D004277:DNA, Single-Stranded / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D014498:Uracil / Q000378:metabolism*
chemical_list: D002457:Cell Extracts; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; C504091:NABP2 protein, human; D014498:Uracil; D004247:DNA; C476001:ABHD1 protein, human; D002265:Carboxylic Ester Hydrolases; C492570:CCNO protein, human; D045647:DNA Glycosylases; C473634:NEIL1 protein, human; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase