Physiological and Biochemical Defects in Functional Interactions of Mitochondrial DNA Polymerase and DNA-binding Mutants of Single-stranded DNA-binding Protein

Abstract

Mitochondrial biogenesis is a key process in animal cell proliferation, and mtDNA replication is an essential component of that process. Whereas a large number of proteins participate in multi-component replication machines in bacterial and nuclear systems, a minimal set of essential proteins are likely involved in the mtDNA replication process (1). We have demonstrated functional interactions between the two-subunit Drosophila mitochondrial DNA polymerase (pol ␥) 1 and the homotetrameric mitochondrial single-stranded DNA-binding protein, mtSSB, at the levels of template-primer binding and initiation and elongation of DNA strand synthesis (2). These studies have documented roles for mtSSB in enhancing primer recognition and binding and in stimulation by 20-to 30-fold of the rate of initiation of DNA strands by pol ␥. Moreover, mtSSB increases severalfold the processivity of pol ␥ in DNA strand elongation.To probe further the biochemical and physiological importance of these functional interactions, we have evaluated the effects of altered forms of mtSSB on DNA binding per se and upon the coordinated reactions involving mtSSB and pol ␥. We find that even modest DNA-binding defects in mtSSB substantially affect DNA synthesis by pol ␥. Furthermore, under conditions that reduce cellular levels of endogenous wild-type mtSSB, a mtDNA depletion phenotype develops that is rescued by production of exogenous wild-type but not mutant mtSSB. EXPERIMENTAL PROCEDURESNucleotides and Nucleic Acids-Unlabeled deoxy-and ribonucleotides were purchased from Amersham Biosciences. [ 3 H]dTTP, [␣-32 P]dATP, and [␥-32 P]ATP were purchased from ICN Biochemicals. Wild-type (6,407 nucleotides (nt)) and recombinant (10,650 nt) M13 DNAs were prepared by standard laboratory methods. Oligodeoxynucleotides complementary to the M13 viral DNAs were synthesized in an Applied Biosystems oligonucleotide synthesizer. The primer for the recombinant M13 DNA that was used in the pol ␥ stimulation assay was 17 nt in length and the primer for the M13 wild-type DNA used in the processivity analysis was 15 nt in length. To prepare template primers for DNA synthesis, M13 DNAs were added to the 15-and 17-mer oligonucleotide primers to a concentration of ϳ70 mM (as nt, in 4-fold molar excess over homologous oligonucleotide), and the DNA mixtures were precipitated with ethanol. The pellets were resuspended in a buffer (0.1 ml) containing 10 mM Tris-HCl, pH 8.0, 0.3 M NaCl, and 0.03 M sodium citrate and were incubated at 65°C for 1 h, followed by incubation at 37°C for an additional hour to anneal the primer to the template. The sequence of the 38-mer oligonucleotide used in the DNA binding and gel mobility shift assays (GMSA) is complementary to positions 6291-6329 in M13mp7 DNA.Enzymes and Proteins-Drosophila DNA polymerase ␥ (Fraction VI, Ͼ90% homogeneous) was prepared from embryonic mitochondria, as described by Wernette and Kaguni (3). Recombinant Drosophila mtSSB (Ͼ90% homogeneous) was prepared as described by Farr et al. (2). T4 polynucleotide kinase and ...

Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA

“…Together with mitochondrial DNA helicase (also known as Twinkle) and the mitochondrial replicase, Pol ␥, it reconstitutes a minimal replisome for human mitochondria (2). Knockdown of mtSSB in human HeLa cells results in reduction of mitochondrial DNA (mtDNA) copy number due to a decrease in mtDNA synthesis (3), as was reported earlier for Drosophila Schneider cells in culture (4). Moreover, lopo mutants of Drosophila melanogaster in which mtSSB is absent die at an early stage in development, concomitant with the complete loss of mtDNA and respiratory capacity (5).…”

“…The presence of mtSSB 4 is a hallmark of replicating mitochondrial nucleoids (1). Together with mitochondrial DNA helicase (also known as Twinkle) and the mitochondrial replicase, Pol ␥, it reconstitutes a minimal replisome for human mitochondria (2).…”

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

“…In addition, human mtSSB (HsmtSSB) stimulates stranddisplacement DNA synthesis promoted by the combined action of the human pol ␥ (Hspol ␥) and HsmtDNA helicase (2). In vivo, absence or depletion of mtSSB protein causes reduction of mtDNA copy number in Drosophila and human cells in culture (6,7), and lethality at the third larval stage in developing D. melanogaster (8). Although mtSSB mutations have not thus far been documented in association with human diseases, as is the case for the genes encoding pol ␥ and mtDNA helicase, a recent report shows a correlation between the levels of mtSSB protein, mtDNA copy number and the aggressiveness of human osteosarcoma cells, suggesting a link between mtDNA replication and cancer progression (9).…”

Knockdown of Mitochondrial Heat Shock Protein 70 Promotes Progeria-like Phenotypes in Caenorhabditis elegans

“…Mitochondria also play essential roles in controlling cell viability and proliferation (1,2). A large number of studies have shown the essential roles of mitochondria in development and differentiation (3,4).…”