The accessory subunit of human DNA polymerase γ is required for mitochondrial DNA maintenance and is able to stabilize the catalytic subunit

Do, Yura; Matsuda, Shigeru; Inatomi, Teppei; Nakada, Kazuto; Yasukawa, Takehiro; Kang, Dongchon

doi:10.1016/j.mito.2020.05.008

Cited by 7 publications

(10 citation statements)

References 37 publications

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“…S3C). These results suggest that Tfam and Polg2 are required for Polg stability at the protein level, consistent with a recent report that Polg2 KO or knockdown in vitro leads to reductions in Polg and Tfam (26). We also observed that Tfam-KO hepatocytes downregulated…”

Section: Mice Tolerate Transient Loss Of Mitochondrial Dna In Hepatoc...supporting

confidence: 93%

Rapid in vivo multiplexed editing (RIME) of the adult mouse liver

Katsuda

Cure

Simeonov

et al. 2022

Preprint

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Background & AimsAssessing mammalian gene function in vivo has traditionally relied on manipulation of the mouse genome in embryonic stem cells or peri-zygotic embryos. These approaches are time consuming and require extensive breeding when simultaneous mutations in multiple genes is desired. The aim of this study is to introduce a Rapid In vivoMultiplexed Editing (RIME), and to provide a proof-of-concept of this system.Approach & ResultsRIME, a system wherein CRISPR/Cas9 technology, paired with adeno-associated viruses (AAVs), permits the inactivation of one or more genes in the adult mouse liver. The method is quick, requiring as little as 1 month from conceptualization to knockout (KO), and highly efficient, enabling editing in >95% of target cells. To highlight its utility, we used this system to inactivate, alone or in combination, genes with functions spanning metabolism, mitosis, mitochondrial maintenance, and cell proliferation.ConclusionRIME enables the rapid, efficient, and inexpensive analysis of multiple genes in the mouse liver in vivo.

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Section: Mice Tolerate Transient Loss Of Mitochondrial Dna In Hepatoc...supporting

confidence: 93%

Rapid in vivo multiplexed editing (RIME) of the adult mouse liver

Katsuda

Cure

Simeonov

et al. 2022

Preprint

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“…These data suggest that POLγA WT is also partially unstable in the absence of POLγB. Accordingly, a recent report demonstrated that human POLγB-knockout cells showed severe decrease in POLγA levels ( 38 ).…”

Section: Resultsmentioning

confidence: 90%

DNA polymerase gamma mutations that impair holoenzyme stability cause catalytic subunit depletion

Silva-Pinheiro

Pardo-Hernández

Reyes

et al. 2021

Nucleic Acids Research

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Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generate the PolgA449T/A449T mouse model, which reproduces the A467T change, the most common human recessive mutation of POLG. We show that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interactions with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues. Therefore, in addition to its role as a processivity factor, POLγB acts to stabilize POLγA and to prevent LONP1-dependent degradation. Notably, we validated this mechanism for other disease-associated mutations affecting the interaction between the two POLγ subunits. We suggest that targeting POLγA turnover can be exploited as a target for the development of future therapies.

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“…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%

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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The accessory subunit of human DNA polymerase γ is required for mitochondrial DNA maintenance and is able to stabilize the catalytic subunit

Cited by 7 publications

References 37 publications

Rapid in vivo multiplexed editing (RIME) of the adult mouse liver

Rapid in vivo multiplexed editing (RIME) of the adult mouse liver

DNA polymerase gamma mutations that impair holoenzyme stability cause catalytic subunit depletion

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

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