Physiological and biochemical defects in functional interactions of mitochondrial DNA polymerase and DNA-binding mutants of single-stranded DNA-binding protein.
Functional interactions between mitochondrial DNA polymerase (pol gamma) and mitochondrial single-stranded DNA-binding protein (mtSSB) from Drosophila embryos greatly enhance the overall activity of pol gamma by increasing primer recognition and binding and stimulating the rate of initiation of DNA strands (Farr, C. L., Wang, Y., and Kaguni, L. S. (1999) J. Biol. Chem. 274, 14779-14785). We show here that DNA-binding mutants of mtSSB are defective in stimulation of DNA synthesis by pol gamma. RNAi knock-down of mtSSB reduces expression to <5% of its normal level in Schneider cells, resulting in growth defects and in the depletion of mitochondrial DNA (mtDNA). Overexpression of mtSSB restores cell growth rate and the copy number of mtDNA, whereas overexpression of a DNA-binding and functionally impaired form of mtSSB neither rescues the cell growth defect nor the mtDNA depletion phenotype. Further development of Drosophila animal models, in which induced mtDNA depletion is manipulated by controlling exogenous expression of wild-type or mutant forms, will offer new insight into the mechanism and progression of human mtDNA depletion syndromes and possible intervention schemes.
Farr
Carol L
CL
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319, USA.
Matsushima
Yuichi
Y
Lagina
Anthony T
AT
Luo
Ningguang
N
Kaguni
Laurie S
LS
D020413:3' Untranslated Regions; D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D000074002:DNA Polymerase gamma; D004268:DNA-Binding Proteins / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D034622:RNA Interference; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
D020413:3' Untranslated Regions; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004247:DNA; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase