title: Reduced stimulation of recombinant DNA polymerase γ and mitochondrial DNA (mtDNA) helicase by variants of mitochondrial single-stranded DNA-binding protein (mtSSB) correlates with defects in mtDNA replication in animal cells.
abstract: The mitochondrial single-stranded DNA-binding protein (mtSSB) is believed to coordinate the functions of DNA polymerase γ (pol γ) and the mitochondrial DNA (mtDNA) helicase at the mtDNA replication fork. We generated five variants of the human mtSSB bearing mutations in amino acid residues specific to metazoans that map on the protein surface, removed from the single-stranded DNA (ssDNA) binding groove. Although the mtSSB variants bound ssDNA with only slightly different affinities, they exhibited distinct capacities to stimulate the DNA polymerase activity of human pol γ and the DNA unwinding activity of human mtDNA helicase in vitro. Interestingly, we observed that the variants with defects in stimulating pol γ had unaltered capacities to stimulate the mtDNA helicase; at the same time, variants showing reduced stimulation of the mtDNA helicase activity promoted DNA synthesis by pol γ similarly to the wild-type mtSSB. The overexpression of the equivalent variants of Drosophila melanogaster mtSSB in S2 cells in culture caused mtDNA depletion under conditions of mitochondrial homeostasis. Furthermore, we observed more severe reduction of mtDNA copy number upon expression of these proteins during recovery from treatment with ethidium bromide, when mtDNA replication is stimulated in vivo. Our findings suggest that mtSSB uses distinct structural elements to interact functionally with its mtDNA replisome partners and to promote proper mtDNA replication in animal cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Mitochondrial Science and Medicine, and Graduate Program in Genetics, Michigan State University, East Lansing, Michigan 48824-1319, USA.
authors.forename: Marcos T
authors.initials: MT
authors.lastname: Oliveira
authors.forename: Laurie S
authors.initials: LS
authors.lastname: Kaguni
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D004265:DNA Helicases / Q000378:metabolism*; D000074002:DNA Polymerase gamma; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology; D006706:Homeostasis / Q000235:genetics; D006801:Humans; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D024101:Mitochondrial Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism*; D013045:Species Specificity
chemical_list: D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; C504091:NABP2 protein, human; C569294:mtSSB protein, Drosophila; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases