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

ABSTRACT: 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.

SUBMITTER: Kozhukhar N

PROVIDER: S-EPMC9316749 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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

Kozhukhar Natalya N Spadafora Domenico D Rodriguez Yelitza A R YAR Alexeyev Mikhail F MF

Cells 20220711 14

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) sub ...[more]

PMID: 35883613

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

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

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