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Reduced stimulation of recombinant DNA polymerase ? 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 ? (pol ?) 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 ? and the DNA unwinding activity of human mtDNA helicase in vitro. Interestingly, we observed that the variants with defects in stimulating pol ? 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 ? 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.

SUBMITTER: Oliveira MT

PROVIDER: S-EPMC3220507 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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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.

Oliveira Marcos T MT Kaguni Laurie S LS

The Journal of biological chemistry 20110926 47

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 exhibit ...[more]

PMID: 21953457

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Project description:BackgroundIn muscle cytochrome oxidase (COX) negative fibers (mitochondrial mosaics) have often been visualized.MethodsCOX activity staining of liver for light and electron microscopy, muscle stains, blue native gel electrophoresis and activity assays of respiratory chain proteins, their immunolocalisation, mitochondrial and nuclear DNA analysis.ResultsThree unrelated infants showed a mitochondrial mosaic in the liver after staining for COX activity, i.e. hepatocytes with strongly reactive mitochondria were found adjacent to cells with many negative, or barely reactive, mitochondria. Deficiency was most severe in the patient diagnosed with Pearson syndrome. Ragged-red fibers were absent in muscle biopsies of all patients. Enzyme biochemistry was not diagnostic in muscle, fibroblasts and lymphocytes. Blue native gel electrophoresis of liver tissue, but not of muscle, demonstrated a decreased activity of complex IV; in both muscle and liver subcomplexes of complex V were seen. Immunocytochemistry of complex IV confirmed the mosaic pattern in two livers, but not in fibroblasts. MRI of the brain revealed severe white matter cavitation in the Pearson case, but only slight cortical atrophy in the Alpers-Huttenlocher patient, and a normal image in the 3rd. MtDNA in leucocytes showed a common deletion in 50% of the mtDNA molecules of the Pearson patient. In the patient diagnosed with Alpers-Huttenlocher syndrome, mtDNA was depleted for 60% in muscle. In the 3rd patient muscular and hepatic mtDNA was depleted for more than 70%. Mutations in the nuclear encoded gene of POLG were subsequently found in both the 2nd and 3rd patients.ConclusionHistoenzymatic COX staining of a liver biopsy is fast and yields crucial data about the pathogenesis; it indicates whether mtDNA should be assayed. Each time a mitochondrial disorder is suspected and muscle data are non-diagnostic, a liver biopsy should be recommended. Mosaics are probably more frequent than observed until now. A novel pathogenic mutation in POLG is reported.Tentative explanations for the mitochondrial mosaics are, in one patient, unequal partition of mutated mitochondria during mitoses, and in two others, an interaction between products of several genes required for mtDNA maintenance.

Dataset Information

Reduced stimulation of recombinant DNA polymerase ? and mitochondrial DNA (mtDNA) helicase by variants of mitochondrial single-stranded DNA-binding protein (mtSSB) correlates with defects in mtDNA replication in animal cells.

Publications

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.

Similar Datasets

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