Human Mitochondrial DNA Polymerase Holoenzyme: Reconstitution and Characterization

Johnson, Allison A.; Tsai, Yu‐Chih; Graves, and Steven W.; Johnson, Kenneth A.

doi:10.1021/bi992104w

Cited by 133 publications

(171 citation statements)

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“…We used K m dynamics to model these four rates, with a Vmax and Km value for each deoxynucleotide substrate determined on the basis of data reported in the literature (Table 2). Experiments show that polymerase-␥ has ϳ50% the rate of polymerization on double-stranded DNA as it does on the short, overhanging DNA template with which the kinetic values were measured (34,35,43), and that adjustment is made to these V max values (Table 3).…”

Section: Methodsmentioning

confidence: 99%

A computational model of mitochondrial deoxynucleotide metabolism and DNA replication

Bradshaw¹,

Samuels²

2005

American Journal of Physiology-Cell Physiology

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The model includes the transport of deoxynucleosides and deoxynucleotides into the mitochondrial matrix space, as well as their phosphorylation and polymerization into mtDNA. Different simulated cell types (cancer, rapidly dividing, slowly dividing, and postmitotic cells) are represented in this model by different cytoplasmic deoxynucleotide concentrations. We calculated the changes in deoxynucleotide concentrations within the mitochondrion during the course of a mtDNA replication event and the time required for mtDNA replication in the different cell types. On the basis of the model, we define three steady states of mitochondrial deoxynucleotide metabolism: the phosphorylating state (the net import of deoxynucleosides and export of phosphorylated deoxynucleotides), the desphosphorylating state (the reverse of the phosphorylating state), and the efficient state (the net import of both deoxynucleosides and deoxynucleotides). We present five testable hypotheses based on this simulation. First, the deoxynucleotide pools within a mitochondrion are sufficient to support only a small fraction of even a single mtDNA replication event. Second, the mtDNA replication time in postmitotic cells is much longer than that in rapidly dividing cells. Third, mitochondria in dividing cells are net sinks of cytoplasmic deoxynucleotides, while mitochondria in postmitotic cells are net sources. Fourth, the deoxynucleotide carrier exerts the most control over the mtDNA replication rate in rapidly dividing cells, but in postmitotic cells, the NDPK and TK2 enzymes have the most control. Fifth, following from the previous hypothesis, rapidly dividing cells derive almost all of their mtDNA precursors from the cytoplasmic deoxynucleotides, not from phosphorylation within the mitochondrion. simulation; nucleotide phosphorylation; nucleoside transport; mitochondrial DNA THE HUMAN MITOCHONDRIAL GENOME is a circular molecule encoding 13 peptides, 22 tRNAs, and 2 rRNAs. The mammalian mitochondrial genome is highly compact, consisting of only ϳ17,000 bp. Even though mammalian cells typically contain thousands of copies of the mitochondrial genome, Ͻ1% of total cellular DNA is contained inside the mitochondria.Mitochondrial DNA (mtDNA) replication occurs during all phases of the cell cycle, even in postmitotic cells in which nuclear DNA synthesis has ceased. The measured half-life of mtDNA in mammalian cells is 10 -30 days (25). Without this ongoing process of mtDNA replication and the nucleotide metabolism supporting replication, mtDNA depletion occurs rapidly, eventually disrupting the production of ATP and causing failure of the cell to function. Errors in the mitochondrial nucleotide metabolism also can be a cause of mtDNA mutations by changing the balance of the four deoxynucleotide triphosphate pools in the mitochondrion (72). Many genetic diseases characterized by mtDNA depletion or increased mtDNA mutation rates are caused by mutations in the nuclear genes for enzymes of nucleotide metabolism (48). To understand these diseases, we must fi...

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Section: Methodsmentioning

confidence: 99%

A computational model of mitochondrial deoxynucleotide metabolism and DNA replication

Bradshaw¹,

Samuels²

2005

American Journal of Physiology-Cell Physiology

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“…1 and 2). The 55-kDa accessory subunit (p55) confers processive DNA synthesis and tight binding of the pol ␥ complex to DNA (4,5).…”

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confidence: 99%

Disease Mutations in the Human Mitochondrial DNA Polymerase Thumb Subdomain Impart Severe Defects in Mitochondrial DNA Replication

Kasiviswanathan

Longley

Chan³

et al. 2009

Journal of Biological Chemistry

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Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol ␥), cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol ␥ revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain (G848S, c.2542g3a; T851A, c.2551a3g; R852C, c.2554c3t; R853Q, c.2558g3a) and two Alpers mutations at less conserved positions in the adjacent palm subdomain (Q879H, c.2637g3t and T885S, c.2653a3t). Biochemical characterization of purified, recombinant forms of pol ␥ revealed that Alpers mutations in the thumb subdomain reduced polymerase activity more than 99% relative to the wild-type enzyme, whereas the palm subdomain mutations retained 50 -70% wildtype polymerase activity. All six mutant enzymes retained physical and functional interaction with the pol ␥ accessory subunit (p55), and none of the six mutants exhibited defects in misinsertion fidelity in vitro. However, differential DNA binding by these mutants suggests a possible orientation of the DNA with respect to the polymerase during catalysis. To our knowledge this study represents the first structure-function analysis of the thumb subdomain in pol ␥ and examines the consequences of mitochondrial disease mutations in this region.As the only DNA polymerase found in animal cell mitochondria, DNA polymerase ␥ (pol ␥) 3 bears sole responsibility for DNA synthesis in all replication and repair transactions involving mitochondrial DNA (1, 2). Mammalian cell pol ␥ is a heterotrimeric complex composed of one catalytic subunit of 140 kDa (p140) and two 55-kDa accessory subunits (p55) that form a dimer (3). The catalytic subunit contains an N-terminal exonuclease domain connected by a linker region to a C-terminal polymerase domain. Whereas the exonuclease domain contains essential motifs I, II, and III for its activity, the polymerase domain comprising the thumb, palm, and finger subdomains contains motifs A, B, and C that are crucial for polymerase activity. The catalytic subunit is a family A DNA polymerase that includes bacterial pol I and T7 DNA polymerase and possesses DNA polymerase, 3Ј 3 5Ј exonuclease, and 5Ј-deoxyribose phosphate lyase activities (for review, see Refs. 1 and 2). The 55-kDa accessory subunit (p55) confers processive DNA synthesis and tight binding of the pol ␥ complex to DNA (4, 5).Depletion of mtDNA as well as the accumulation of deletions and point mutations in mtDNA have been observed in several mitochondrial disorders (for review, see Ref. 6). mtDNA depletion syndromes are caused by defects in nuclear genes responsible for replication and maintenance of the mitochondrial genome (7). Mutation of POLG, the gene encoding the catalytic subunit of pol ␥, is frequently involved in disorders linked to mutagenesis of mtDNA (8, 9). Presently, more than 150 point mutations in POLG are linked with a wide variety of mitochondrial diseases, including the autosomal dominant (ad) and...

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“…and purification of the catalytic subunit (pol ␥A) and the accessory subunit (pol ␥B) were accomplished as described previously (7,8). An exonuclease-deficient mutant of the catalytic subunit (E200A), pol ␥A exo Ϫ , was purified as described (9); this mutation reduced the exonuclease rate greater than 10 7 -fold without affecting the kinetic parameters governing polymerization.…”

Section: Expression and Purification Of Enzyme Subunits-expressionmentioning

confidence: 99%

Fidelity and Processivity of Reverse Transcription by the Human Mitochondrial DNA Polymerase

Lee

Johnson

2007

Journal of Biological Chemistry

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We have characterized, by transient-state kinetic methods, the polymerase and exonuclease activities of the human mitochondrial DNA polymerase (pol ␥) during reverse transcription, employing a synthetic oligonucleotide consisting of a DNA primer and an RNA template. In comparison with the kinetic parameters observed with a DNA template, the rate of correct deoxynucleotide incorporation was reduced 25-fold (5.5 ؎ 0.2 s ؊1 ), whereas the dissociation constant (K d ) for nucleotide binding was increased 4-fold (12 ؎ 1 M). In addition, discrimination against mismatches was reduced ϳ20-fold to only 15,000 on average. The proofreading exonuclease favored the removal of an incorrect nucleotide (0.0021 ؎ 0.0002 s ؊1 for correct versus 0.034 ؎ 0.004 s ؊1 for incorrect), and the partitioning between incorporation beyond a mismatch (5.5 ؋ 10 ؊5 ؎ 0.4 ؋ 10 ؊5 s ؊1 ), and exonuclease removal of that mismatch favors removal of the mismatch. These data suggest that the "reverse transcriptase activity" of mitochondrial polymerase could be physiologically relevant. However, the enzyme stalls and is unable to efficiently incorporate beyond a single nucleotide with an RNA template. Additionally, we present a refined method for calculating net discrimination, which more accurately describes the contributions of correct and incorrect incorporation. The biological and biotechnological significance of these results are discussed.Reverse transcription is the process of DNA polymerization using an RNA template, and it is interesting to note that mitochondrial DNA polymerases (pol ␥) were initially characterized by their ability to reverse-transcribe DNA from RNA (1). In fact, over the past 30 years, human pol 2 ␥ has been shown to be an effective reverse transcriptase in steady state single nucleotide incorporation assays using DNA/RNA heteroduplexes to probe for its presence and to assay its replication fidelity (1-4). RNase digestion of mammalian mitochondrial DNA has shown that there are at least 30 ribonucleotides incorporated in the mitochondrial genome (5), but the origin and significance of these ribonucleotides are unknown. They could be the products of incorporation by the DNA polymerase or merely remnants of RNA priming by the mitochondrial RNA polymerase after lagging strand synthesis and incomplete primer removal.Steady state assays have been used to characterize reverse transcriptase activity and fidelity of pol ␥ largely because steady state rates are faster with an RNA template. However, steady state, single nucleotide turnover rates are limited by polymerase dissociation from product complexes and do not provide reliable measures of nucleotide discrimination. Therefore, presteady state kinetic approaches have been used to begin to characterize more accurately the reverse transcriptase activity of human pol ␥ (6). In our previous studies we were surprised to see how rapidly pol ␥ catalyzed a single DNA incorporation with an RNA template. Here we demonstrate a marked reduction in fidelity during reverse transcription and s...

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Human Mitochondrial DNA Polymerase Holoenzyme: Reconstitution and Characterization

Cited by 133 publications

References 26 publications

A computational model of mitochondrial deoxynucleotide metabolism and DNA replication

A computational model of mitochondrial deoxynucleotide metabolism and DNA replication

Disease Mutations in the Human Mitochondrial DNA Polymerase Thumb Subdomain Impart Severe Defects in Mitochondrial DNA Replication

Fidelity and Processivity of Reverse Transcription by the Human Mitochondrial DNA Polymerase

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