Laurie S. Kaguni

Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM)

Lon is the major protease in the mitochondrial matrix in eukaryotes, and is well conserved among species. Although a role for Lon in mitochondrial biogenesis has been proposed, the mechanistic basis is unclear. Here, we demonstrate a role for Lon in mtDNA metabolism. An RNA interference (RNAi) construct was designed that reduces Lon to less than 10% of its normal level in Drosophila Schneider cells. RNAi knockdown of Lon results in increased abundance of mitochondrial transcription factor A (TFAM) and mtDNA copy number. In a corollary manner, overexpression of Lon reduces TFAM levels and mtDNA copy number. Notably, induction of mtDNA depletion in Lon knockdown cells does not result in degradation of TFAM, thereby causing a dramatic increase in the TFAM∶mtDNA ratio. The increased TFAM∶mtDNA ratio in turn causes inhibition of mitochondrial transcription. We conclude that Lon regulates mitochondrial transcription by stabilizing the mitochondrial TFAM∶mtDNA ratio via selective degradation of TFAM.

Functional interactions of mitochondrial DNA polymerase and single-stranded DNA-binding protein. Template-primer DNA binding and initiation and elongation of DNA strand synthesis.

Functional interactions between mitochondrial DNA polymerase (pol γ) and mitochondrial single-stranded DNA-binding protein (mtSSB) from Drosophila embryos have been evaluated with regard to the overall activity of pol γ and in partial reactions involving template-primer binding and initiation and idling in DNA strand synthesis. Both the 5′ → 3′ DNA polymerase and 3′ → 5′ exonuclease in pol γ are stimulated 15–20-fold on oligonucleotide-primed single-stranded DNA by native and recombinant forms of mtSSB. That the extent of stimulation is similar for both enzyme activities over a broad range of KCl concentrations suggests their functional coordination and a similar mechanism of stimulation by mtSSB. At the same time, the high mispair specificity of pol γ in exonucleolytic hydrolysis is maintained, indicating that enhancement of pol γ catalytic efficiency is likely not accompanied by increased nucleotide turnover. DNase I footprinting of pol γ·DNA complexes and initial rate measurements show that mtSSB enhances primer recognition and binding and stimulates 30-fold the rate of initiation of DNA strands. Dissociation studies show that productive complexes of the native pol γ heterodimer with template-primer DNA are formed and remain stable in the absence of replication accessory proteins.

Drosophila mitochondrial transcription factor B1 modulates mitochondrial translation but not transcription or DNA copy number in Schneider cells.

We report the cloning and molecular analysis of Drosophila mitochondrial transcription factor (d-mtTF) B1. An RNA interference (RNAi) construct was designed that reduces expression of d-mtTFB1 to 5% of its normal level in Schneider cells. In striking contrast with our previous study on d-mtTFB2, we found that RNAi knock-down of d-mtTFB1 does not change the abundance of specific mitochondrial RNA transcripts, nor does it affect the copy number of mitochondrial DNA. In a corollary manner, overexpression of d-mtTFB1 did not increase either the abundance of mitochondrial RNA transcripts or mitochondrial DNA copy number. Our data suggest that, unlike d-mtTFB2, d-mtTFB1 does not have a critical role in either transcription or regulation of the copy number of mitochondrial DNA. Instead, because we found that RNAi knockdown of d-mtTFB1 reduces mitochondrial protein synthesis, we propose that it serves its primary role in modulating translation. Our work represents the first study to document the role of mtTFB1 in vivo and establishes clearly functional differences between mtTFB1 and mtTFB2.

Accessory subunit of mitochondrial DNA polymerase from Drosophila embryos. Cloning, molecular analysis, and association in the native enzyme.

A full-length cDNA of the accessory (β) subunit of mitochondrial DNA polymerase from Drosophilaembryos has been obtained, and its nucleotide sequence was determined. The cDNA clone encodes a polypeptide with a deduced amino acid sequence of 361 residues and a predicted molecular mass of 41 kDa. The gene encoding the β subunit lies within 4 kilobase pairs of that for the catalytic subunit in the Drosophila genome, on the left arm of chromosome 2. The two genes have similar structural features and share several common DNA sequence elements in their upstream regions, suggesting the possibility of coordinate regulation. A human cDNA homolog of the accessory subunit was identified, and its nucleotide sequence was determined. The human sequence encodes a polypeptide with a predicted molecular mass of 43 kDa that shows a high degree of amino acid sequence similarity to the Drosophila β subunit. Subunit-specific rabbit antisera, directed against the recombinant catalytic and accessory subunit polypeptides overexpressed and purified from Escherichia coli, recognize specifically and immunoprecipitate the native enzyme from Drosophilaembryos. Demonstration of the physical association of the two subunits in the Drosophila enzyme and identification of a human accessory subunit homolog provide evidence for a common heterodimeric structure for animal mitochondrial DNA polymerases.

Mitochondrial Single-stranded DNA-binding Protein from Drosophila Embryos PHYSICAL AND BIOCHEMICAL CHARACTERIZATION

Using a stringent purification procedure on single-stranded DNA cellulose, we have isolated the mitochondrial single-stranded DNA-binding protein from Drosophila melanogaster embryos. Its identity is demonstrated by amino-terminal sequencing of the homogeneous protein and by its localization to a mitochondrial protein fraction. The mitochondrial protein is immunologically and biochemically distinct from the previously characterized nuclear replication protein A from Drosophila (Mitsis, P. G., Kowalczykowski, S. C., and Lehman, I. R.(1993) Biochemistry 32, 5257-5266; Marton, R. F., Thömmes, P., and Cotterill, S.(1994) FEBS Lett. 342, 139-144). It consists of a single polypeptide of 18 kDa, which is responsible for the DNA binding activity. Sedimentation analysis suggests that D. melanogaster mitochondrial single-stranded DNA-binding protein exists as a homo-oligomer, possibly a tetramer, in solution. The protein binds to DNA in its single-stranded form with a strong preference over double-stranded DNA or RNA, and binds to polypyrimidines preferentially over polypurines. Drosophila mitochondrial single-stranded DNA-binding protein exhibits a greater affinity for long oligonucleotides as compared to short ones, yet does not show high cooperativity. Its binding site size, determined by competition studies and by fluorescence quenching, is approximately 17 nucleotides under low salt conditions, and increases in the presence of greater than 150 mM NaCl. The homogeneous protein stimulates the activity of mitochondrial DNA polymerase from D. melanogaster embryos, increasing dramatically the rate of initiation of DNA synthesis on a singly primed DNA template.

The accessory subunit of DNA polymerase γ is essential for mitochondrial DNA maintenance and development in Drosophila melanogaster

DNA polymerase γ, Pol γ, is the key replicative enzyme in animal mitochondria. The Drosophila enzyme is a heterodimer comprising catalytic and accessory subunits of 125 kDa and 35 kDa, respectively. Both subunits have been cloned and characterized in a variety of model systems, and genetic mutants of the catalytic subunit were first identified in Drosophila, as chemically induced mutations that disrupt larval behavior (tamas). Mutations in the gene encoding the accessory subunit have not yet been described in any organism. Here, we report the consequences of null mutations upon mitochondrial DNA (mtDNA) replication and morphology, cell proliferation, and organismal viability. Mutations in the accessory subunit cause lethality during early pupation, concomitant with loss of mtDNA and mitochondrial mass, and reduced cell proliferation in the central nervous system. Surprisingly, the function of the central nervous system and muscle, as assessed in a locomotion assay, are only marginally affected. This finding is in contrast to our previous findings that disruption in the function of the catalytic subunit causes severe reduction in larval locomotion. We discuss our results in the context of current hypotheses for the function of the accessory subunit in mtDNA replication.

Subunit Structure of Mitochondrial DNA Polymerase from Drosophila Embryos PHYSICAL AND IMMUNOLOGICAL STUDIES

The subunit structure of mitochondrial DNA polymerase from Drosophila embryos has been examined by a combination of physical and immunological methods. A highly specific rabbit antiserum directed against the native enzyme was developed and found to recognize specifically its two subunits in immunoblot and immunoprecipitation analyses. That and the potent inhibition by the rabbit antiserum of the DNA polymerase and 3′ 5′ exonuclease activities of the nearly homogeneous mitochondrial DNA polymerase provide strong evidence for the physical association of the 3′ 5′ exonuclease with the two subunit enzyme. An immunoprecipitation analysis of crude enzyme fractions showed that the two subunits of Drosophila mitochondrial DNA polymerase are intact, and an in situ gel proteolysis analysis showed that they are structurally distinct. Template-primer DNA binding studies demonstrated formation of a stable and discrete enzyme-DNA complex in the absence of accessory proteins. Photochemical cross-linking of the complexes by UV light indicated that the α but not the β subunit of mitochondrial DNA polymerase makes close contact with DNA, and limited digestion of the native enzyme with trypsin showed that an 65-kDa proteolytic fragment of the α subunit retains the DNA binding function.

Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

We report the cloning and molecular analysis of Drosophila mitochondrial DNA helicase (d-mtDNA helicase) homologous to human TWINKLE, which encodes one of the genes responsible for autosomal dominant progressive external ophthalmoplegia. An RNA interference construct was designed that reduces expression of d-mtDNA helicase to an undetectable level in Schneider cells. RNA interference knockdown of d-mtDNA helicase decreases the copy number of mitochondrial DNA (mtDNA) ∼5-fold. In a corollary manner, overexpression of d-mtDNA helicase increases mtDNA levels 1.4-fold. Overexpression of helicase active site mutants K388A and D483A results in a severe depletion of mtDNA and a dominant negative lethal phenotype. Overexpression of mutants analogous to human autosomal dominant progressive external ophthalmoplegia mutations shows differential effects. Overexpression of I334T and A442P mutants yields a dominant negative effect as for the active site mutants. In contrast, overexpression of A326T, R341Q, and W441C mutants results in increased mtDNA copy number, as observed with wild-type overexpression. Our dominant negative analysis of d-mtDNA helicase in cultured cells provides a tractable model for understanding human autosomal dominant progressive external ophthalmoplegia mutations.

Differential Regulation of the Catalytic and Accessory Subunit Genes of Drosophila Mitochondrial DNA Polymerase

The developmental pattern of expression of the genes encoding the catalytic (α) and accessory (β) subunits of mitochondrial DNA polymerase (pol γ) has been examined inDrosophila melanogaster. The steady-state level of pol γ-β mRNA increases during the first hours of development, reaching its maximum value at the start of mtDNA replication inDrosophila embryos. In contrast, the steady-state level of pol γ-α mRNA decreases as development proceeds and is low in stages of active mtDNA replication. This difference in mRNA abundance results at least in part from differences in the rates of mRNA synthesis. The pol γ genes are located in a compact cluster of five genes that contains three promoter regions (P1–P3). The P1 region directs divergent transcription of the pol γ-β gene and the adjacent rpII33 gene. P1 contains a DNA replication-related element (DRE) that is essential for pol γ-β promoter activity, but not for rpII33 promoter activity in Schneiders cells. A second divergent promoter region (P2) controls the expression of theorc5 and sop2 genes. The P2 region contains two DREs that are essential for orc5 promoter activity, but not for sop2 promoter activity. The expression of the pol γ-α gene is directed by P3, a weak promoter that does not contain DREs. Electrophoretic mobility shift experiments demonstrate that the DRE-binding factor (DREF) regulatory protein binds to the DREs in P1 and P2. DREF regulates the expression of several genes encoding key factors involved in nuclear DNA replication. Its role in controlling the expression of the pol γ-β and orc5genes establishes a common regulatory mechanism linking nuclear and mitochondrial DNA replication. Overall, our results suggest that the accessory subunit of mtDNA polymerase plays an important role in the control of mtDNA replication in Drosophila.

Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of the human mitochondrial replicase, Pol γ

Mutations in Pol γ represent a major cause of human mitochondrial diseases, especially those affecting the nervous system in adults and in children. Recessive mutations in Pol γ represent nearly half of those reported to date, and they are nearly uniformly distributed along the length of the POLG1 gene (Human DNA Polymerase gamma Mutation Database); the majority of them are linked to the most severe form of POLG syndrome, Alpers–Huttenlocher syndrome. In this report, we assess the structure–function relationships for recessive disease mutations by reviewing existing biochemical data on site-directed mutagenesis of the human, Drosophila and yeast Pol γs, and their homologs from the family A DNA polymerase group. We do so in the context of a molecular model of Pol γ in complex with primer–template DNA, which we have developed based upon the recently solved crystal structure of the apoenzyme form. We present evidence that recessive mutations cluster within five distinct functional modules in the catalytic core of Pol γ. Our results suggest that cluster prediction can be used as a diagnosis-supporting tool to evaluate the pathogenic role of new Pol γ variants.