TFB2M and POLRMT are essential for mammalian mitochondrial DNA replication

Inatomi, Teppei; Matsuda, Shigeru; Ishiuchi, Takashi; Do, Yura; Nakayama, Masunari; Abe, Shusaku; Kasho, Kazutoshi; Wanrooij, Sjoerd; Nakada, Kazuto; Ichiyanagi, Kenji; Sasaki, Hidetada; Yasukawa, Takehiro; Kang, Dongchon

doi:10.1016/j.bbamcr.2021.119167

Cited by 11 publications

(8 citation statements)

References 54 publications

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“…In this study we demonstrated that the lethality of the knockouts of the critical components of the mtDNA replication apparatus is conditional. Independently, D. Kang's group reached the same conclusion for a subset of proteins examined in this study [12,13]. These observations open opportunities for reverse genetic analysis of proteins involved in mtDNA replication.…”

Section: Discussionsupporting

confidence: 74%

“…As a result, attempts to cultivate KO cells for any critical component of the mtDNA replication apparatus have been unsuccessful until recently. However, while this work was in progress, Kang’s group succeeded in inactivating POLRMT, TFB2M , and POLG2 in cultured cells [ 12 , 13 ].…”

Section: Introductionmentioning

confidence: 99%

“…been unsuccessful until recently. However, while this work was in progress, Kang's group succeeded in inactivating POLRMT, TFB2M, and POLG2 in cultured cells [12,13].…”

Section: Introductionmentioning

confidence: 99%

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A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication

Kozhukhar

Spadafora

Rodriguez

et al. 2022

Cells

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The unavailability of tractable reverse genetic analysis approaches represents an obstacle to a better understanding of mitochondrial DNA replication. Here, we used CRISPR-Cas9 mediated gene editing to establish the conditional viability of knockouts in the key proteins involved in mtDNA replication. This observation prompted us to develop a set of tools for reverse genetic analysis in situ, which we called the GeneSwap approach. The technique was validated by identifying 730 amino acid (aa) substitutions in the mature human TFAM that are conditionally permissive for mtDNA replication. We established that HMG domains of TFAM are functionally independent, which opens opportunities for engineering chimeric TFAMs with customized properties for studies on mtDNA replication, mitochondrial transcription, and respiratory chain function. Finally, we present evidence that the HMG2 domain plays the leading role in TFAM species-specificity, thus indicating a potential pathway for TFAM-mtDNA evolutionary co-adaptations.

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Section: Discussionsupporting

confidence: 74%

Section: Introductionmentioning

confidence: 99%

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A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication

Kozhukhar

Spadafora

Rodriguez

et al. 2022

Cells

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“…For instance, we previously described a role for SSB1 in mitochondrial intron RNA splicing (Qian et al, 2021). Recently, the mitochondrion‐ and chloroplast‐localized phage‐type RNA polymerase RPOTmp was reported to act as a negative regulator of mtDNA replication (Kühn et al, 2009), while dysfunction of its homolog POLRMT in humans resulted in complete mtDNA loss (Inatomi et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

Arabidopsis mitochondrial single‐stranded DNA‐binding proteins SSB1 and SSB2 are essential regulators of mtDNA replication and homologous recombination

Qian

Zheng

Wang

et al. 2022

JIPB

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Faithful DNA replication is one of the most essential processes in almost all living organisms. However, the proteins responsible for organellar DNA replication are still largely unknown in plants.Here, we show that the two mitochondriontargeted single-stranded DNA-binding (SSB) proteins SSB1 and SSB2 directly interact with each other and act as key factors for mitochondrial DNA (mtDNA) maintenance, as their single or double loss-of-function mutants exhibit severe germination delay and growth retardation. The mtDNA levels in mutants lacking SSB1 and/or SSB2 function were two-to four-fold higher than in the wild-type (WT), revealing a negative role for SSB1/ 2 in regulating mtDNA replication. Genetic analysis indicated that SSB1 functions upstream of mitochondrial DNA POLYMERASE IA (POLIA) or POLIB in mtDNA replication, as mutation in either gene restored the high mtDNA copy number of the ssb1-1 mutant back to WT levels. In addition, SSB1 and SSB2 also participate in mitochondrial genome maintenance by influencing mtDNA homologous recombination (HR). Additional genetic analysis suggested that SSB1 functions upstream of ORGANELLAR SINGLE-STRANDED DNA-BINDING PROTEIN1 (OSB1) during mtDNA replication, while SSB1 may act downstream of OSB1 and MUTS HOMOLOG1 for mtDNA HR. Overall, our results yield new insights into the roles of the plant mitochondrion-targeted SSB proteins and OSB1 in maintaining mtDNA stability via affecting DNA replication and DNA HR.

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“…4A). The D-loop region was also enriched but was excluded from our analysis as this area was shown to be prone to artefacts in ChIP-seq protocols, as testified by enrichment upon pull down with IgG (38) or with uniquely localized nuclear transcription factors (39). We next performed mtG4-ChIP followed by qPCR (mtG4-ChIP-qPCR) and amplified two mtDNA regions that are enriched in all three replicates (Fig.…”

Section: Resultsmentioning

confidence: 99%

Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells

Doimo

Abrahamsson

L’Hôte

et al. 2022

Preprint

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Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate.In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrated that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.

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TFB2M and POLRMT are essential for mammalian mitochondrial DNA replication

Cited by 11 publications

References 54 publications

A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication

A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication

Arabidopsis mitochondrial single‐stranded DNA‐binding proteins SSB1 and SSB2 are essential regulators of mtDNA replication and homologous recombination

Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells

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