Effects of DNA lesions on the transcription reaction of mitochondrial RNA polymerase: implications for bypass RNA synthesis on oxidative DNA lesions

Nakanishi, Nozomi; Fukuoh, Atsushi; Kang, Dongchon; Iwai, Shigenori; Kuraoka, Isao

doi:10.1093/mutage/ges060

Cited by 18 publications

(16 citation statements)

References 40 publications

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“…With a deoxyguanosine (dG) at the +50 position (from the LSP start site), POLRMT produced run-off transcripts of 65 nts (Figure 5D, lane 1). In agreement with previously published results (19), a mutation of the dG at +50 to 8-Oxo-dG caused a majority of all POLRMT initiation events to terminate at this site (38% run-off and 62% pre-termination, Figure 5D lane 5 and Figure 5E). When TEFM was added to the reaction, the bypass of the 8-Oxo-dG base was increased to a majority of all initiations (61% run-off and 39% pre-termination, Figure 5D lane 6 and Figure 5E).…”

Section: Resultssupporting

confidence: 93%

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TEFM is a potent stimulator of mitochondrial transcription elongation in vitro

Posse

Shahzad

Falkenberg

et al. 2015

Nucleic Acids Research

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A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest. In the current work, we have isolated active TEFM in recombinant form and used a reconstituted in vitro transcription system to characterize its activities. We show that TEFM strongly promotes POLRMT processivity as it dramatically stimulates the formation of longer transcripts. TEFM also abolishes premature transcription termination at conserved sequence block II, an event that has been linked to primer formation during initiation of mtDNA synthesis. We show that POLRMT pauses at a wide range of sites in a given DNA sequence. In the absence of TEFM, this leads to termination; however, the presence of TEFM abolishes this effect and aids POLRMT in continuation of transcription. Further, we show that TEFM substantially increases the POLRMT affinity to an elongation-like DNA:RNA template. In combination with previously published in vivo observations, our data establish TEFM as an essential component of the mitochondrial transcription machinery.

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Section: Resultssupporting

confidence: 93%

“…Only a small fraction of the transcribing POLRMT molecules manage to pass 8-Oxo-dG sites (19). It is known that in the nucleus, RNAP II can bypass lesions such as 8-Oxo-dG in the presence of an elongation factor, TFIIS (20).…”

Section: Resultsmentioning

confidence: 99%

TEFM is a potent stimulator of mitochondrial transcription elongation in vitro

Posse

Shahzad

Falkenberg

et al. 2015

Nucleic Acids Research

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“…11A) to measure the incorporation of correct CTP and incorrect ATP across 8-oxo-dG. Although elongation studies of POLRMT and T7 RNAP have been conducted with 8-oxo-dG (10,40), the rates of pausing, mutagenic bypass, and error-free bypass have not been measured. We show that POLRMT adds the correct CTP across 8-oxo-dG with a k pol of 9 ϫ 10 Ϫ4 s Ϫ1 (Fig.…”

Section: Effect Of An Oxidized 8-oxo-dg Templating Base On Elongationmentioning

confidence: 99%

Transcriptional fidelities of human mitochondrial POLRMT, yeast mitochondrial Rpo41, and phage T7 single-subunit RNA polymerases

Sultana¹,

Solotchi²,

Ramachandran³

et al. 2017

Journal of Biological Chemistry

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Single-subunit RNA polymerases (RNAPs) are present in phage T7 and in mitochondria of all eukaryotes. This RNAP class plays important roles in biotechnology and cellular energy production, but we know little about its fidelity and error rates. Herein, we report the error rates of three single-subunit RNAPs measured from the catalytic efficiencies of correct and all possible incorrect nucleotides. The average error rates of T7 RNAP (2 × 10), yeast mitochondrial Rpo41 (6 × 10), and human mitochondrial POLRMT (RNA polymerase mitochondrial) (2 × 10) indicate high accuracy/fidelity of RNA synthesis resembling those of replicative DNA polymerases. All three RNAPs exhibit a distinctly high propensity for GTP misincorporation opposite dT, predicting frequent A→G errors in RNA with rates of ∼10 The A→C, G→A, A→U, C→U, G→U, U→C, and U→G errors mostly due to pyrimidine-purine mismatches were relatively frequent (10-10), whereas C→G, U→A, G→C, and C→A errors from purine-purine and pyrimidine-pyrimidine mismatches were rare (10-10). POLRMT also shows a high C→A error rate on 8-oxo-dG templates (∼10). Strikingly, POLRMT shows a high mutagenic bypass rate, which is exacerbated by TEFM (transcription elongation factor mitochondrial). The lifetime of POLRMT on terminally mismatched elongation substrate is increased in the presence of TEFM, which allows POLRMT to efficiently bypass the error and continue with transcription. This investigation of nucleotide selectivity on normal and oxidatively damaged DNA by three single-subunit RNAPs provides the basic information to understand the error rates in mitochondria and, in the case of T7 RNAP, to assess the quality of transcribed RNAs.

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“…The ‘transcriptional mutagenesis’, that is conversion of DNA damage into mutated transcripts by RNA polymerase [27], is a well-established phenomenon. This includes conversion of oxidative damage, for example, 8-OHdG [28] and has been established for various RNA polymerases, including the mitochondrial RNA polymerase [29••]. ‘Transcriptional mutagenesis’ has been recently implicated in cellular stress and premature cellular aging [30••].…”

Section: Hypothesis: Mtdna Damage Contributes To the Mutator Phenotypmentioning

confidence: 99%

Amelioration of premature aging in mtDNA mutator mouse by exercise: the interplay of oxidative stress, PGC-1α, p53, and DNA damage. A hypothesis

Safdar

Annis

Kraytsberg

et al. 2016

Current Opinion in Genetics & Development

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The mtDNA mutator mouse lacks the proofreading capacity of the sole mtDNA polymerase, leading to accumulation of somatic mtDNA mutations, and a profound premature aging phenotype including elevated oxidative stress and apoptosis, and reduced mitochondrial function. We have previously reported that endurance exercise alleviates the aging phenotype in the mutator mice, reduces oxidative stress, and enhances mitochondrial biogenesis. Here we summarize our findings, with the emphasis on the central role of p53 in these adaptations. We demonstrate that mtDNA in sedentary and exercised PolG mice carry similar amounts of mutations in muscle, but in addition to that sedentary mice have more non-mutational damage, which is mitigated by exercise. It follows therefore that the profound alleviation of the mtDNA mutator phenotype in muscle by exercise may not require a reduction in mtDNA mutational load, but rather a decrease of mtDNA damage and/or oxidative stress. We further hypothesize that the observed ‘alleviation without a reduction of mutational load’ implies that the oxidative stress in PolG muscle is maintained, at least in part, by the ‘malicious cycle’, a hypothetical positive feedback potentially driven by the ‘transcriptional mutagenesis’, that is the conversion of chemically modified nucleotides into mutant RNA bases by the mitochondrial RNA polymerase.

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Effects of DNA lesions on the transcription reaction of mitochondrial RNA polymerase: implications for bypass RNA synthesis on oxidative DNA lesions

Cited by 18 publications

References 40 publications

TEFM is a potent stimulator of mitochondrial transcription elongation in vitro

TEFM is a potent stimulator of mitochondrial transcription elongation in vitro

Transcriptional fidelities of human mitochondrial POLRMT, yeast mitochondrial Rpo41, and phage T7 single-subunit RNA polymerases

Amelioration of premature aging in mtDNA mutator mouse by exercise: the interplay of oxidative stress, PGC-1α, p53, and DNA damage. A hypothesis

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