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Double-strand breaks of mouse muscle mtDNA promote large deletions similar to multiple mtDNA deletions in humans.

ABSTRACT: Mitochondrial DNA (mtDNA) deletions are a common cause of mitochondrial disorders and have been found to accumulate during normal aging. Despite the fact that hundreds of deletions have been characterized at the molecular level, their mechanisms of genesis are unknown. We tested the effect of double-strand breaks of muscle mtDNA by developing a mouse model in which a mitochondrially targeted restriction endonuclease (PstI) was expressed in skeletal muscle of mice. Because mouse mtDNA harbors two PstI sites, transgenic founders developed a mitochondrial myopathy associated with mtDNA depletion. The founders showed a chimeric pattern of transgene expression and their residual level of wild-type mtDNA in muscle was approximately 40% of controls. We were able to identify the formation of large mtDNA deletions in muscle of transgenic mice. A family of mtDNA deletions was identified, and most of these rearrangements involved one of the PstI sites and the 3' end of the D-loop region. The deletions had no or small direct repeats at the breakpoint region. These features are essentially identical to the ones observed in humans with multiple mtDNA deletions in muscle, suggesting that double-strand DNA breaks mediate the formation of large mtDNA deletions.

SUBMITTER: Srivastava S

PROVIDER: S-EPMC1242110 | biostudies-literature | 2005 Apr

REPOSITORIES: biostudies-literature

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Double-strand breaks of mouse muscle mtDNA promote large deletions similar to multiple mtDNA deletions in humans.

Srivastava Sarika S Moraes Carlos T CT

Human molecular genetics 20050209 7

Mitochondrial DNA (mtDNA) deletions are a common cause of mitochondrial disorders and have been found to accumulate during normal aging. Despite the fact that hundreds of deletions have been characterized at the molecular level, their mechanisms of genesis are unknown. We tested the effect of double-strand breaks of muscle mtDNA by developing a mouse model in which a mitochondrially targeted restriction endonuclease (PstI) was expressed in skeletal muscle of mice. Because mouse mtDNA harbors two ...[more]

PMID: 15703189

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Project description:Clostridium difficile is an important nosocomial pathogen associated with potentially fatal disease induced by the use of antibiotics. Genetic characterization of such clinically important bacteria is often hampered by lack of availability of suitable tools. Here, we describe the use of I-SceI to induce DNA double-strand breaks, which increase the frequency of allelic exchange and enable the generation of markerless deletions in C. difficile The usefulness of the system is illustrated by the deletion of genes encoding putative AddAB homologues. The ?addAB mutants are sensitive to ultraviolet light and the antibiotic metronidazole, indicating a role in homologous recombination and the repair of DNA breaks. Despite the impairment in recombination, the mutants are still proficient for induction of the SOS response. In addition, deletion of the fliC gene, and subsequent complementation, reveals the importance of potential regulatory elements required for expression of a downstream gene encoding the flagellin glycosyltransferase.IMPORTANCE Most sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution in Clostridium difficile Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions in C. difficile and expands the toolbox available for genetic analysis of this important anaerobic pathogen.

Dataset Information

Double-strand breaks of mouse muscle mtDNA promote large deletions similar to multiple mtDNA deletions in humans.

Publications

Double-strand breaks of mouse muscle mtDNA promote large deletions similar to multiple mtDNA deletions in humans.

Similar Datasets

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