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Analysis of replication intermediates indicates that Drosophila melanogaster mitochondrial DNA replicates by a strand-coupled theta mechanism.

ABSTRACT: Mitochondrial DNA synthesis is necessary for the normal function of the organelle and for the eukaryotic organism as a whole. Here we demonstrate, using two-dimensional agarose gel electrophoresis to analyse replication intermediates, that unidirectional, strand-coupled DNA synthesis is the prevalent mode of mtDNA replication in Drosophila melanogaster. Commencing within the single, extended non-coding region (NCR), replication proceeds around the circular genome, manifesting an irregular rate of elongation, and pausing frequently in specific regions. Evidence for a limited contribution of strand-asynchronous DNA synthesis was found in a subset of mtDNA molecules, but confined to the ribosomal RNA gene region, just downstream of the NCR. Our findings imply that strand-coupled replication is widespread amongst metazoans, and should inform future research on mtDNA metabolism in D. melanogaster.

SUBMITTER: Joers P

PROVIDER: S-EPMC3537619 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Analysis of replication intermediates indicates that Drosophila melanogaster mitochondrial DNA replicates by a strand-coupled theta mechanism.

Jõers Priit P Jacobs Howard T HT

PloS one 20130104 1

Mitochondrial DNA synthesis is necessary for the normal function of the organelle and for the eukaryotic organism as a whole. Here we demonstrate, using two-dimensional agarose gel electrophoresis to analyse replication intermediates, that unidirectional, strand-coupled DNA synthesis is the prevalent mode of mtDNA replication in Drosophila melanogaster. Commencing within the single, extended non-coding region (NCR), replication proceeds around the circular genome, manifesting an irregular rate o ...[more]

PMID: 23308172

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Project description:Mosquito-borne dengue virus (DENV, genus Flavivirus) has emerged as a major threat to global human health in recent decades, and novel strategies to contain the escalating dengue fever pandemic are urgently needed. RNA interference (RNAi) induced by exogenous small interfering RNAs (siRNAs) has shown promise for treatment of flavivirus infections in hosts and prevention of transmission by vectors. However, the impact of RNAi triggered by authentic virus infection on replication of DENV, or any flavivirus, has received little study. The objectives of the current study were threefold: first, to assess the utility of Drosophila melanogaster S2 cells for the study of DENV, second to investigate the impact of multiple enzymes in the RNAi pathway on DENV replication; and third to test for variation in the response of the four serotypes of DENV to modulation of RNAi.Three strains from each of the four DENV serotypes showed replication in S2 cells following infection at multiplicity of infection (MOI) 0.1 and MOI 10; each strain achieved titers > 4.0 log10pfu/ml five days after infection at MOI 10. The four serotypes did not differ in mean titer. S2 cells infected with DENV-1, 2, 3 or 4 produced siRNAs, indicating that infection triggered an RNAi response. Knockdown of one of the major enzymes in the RNAi pathway, Dicer-2 (Dcr-2), resulted in a 10 to 100-fold enhancement of replication of all twelve strains of DENV in S2 cells. While serotypes did not differ in their average response to Dcr-2 knockdown, strains within serotypes showed significant differences in their sensitivity to Dcr-2 knockdown. Moreover, knockdown of three additional components of the RNAi pathway, Argonaute 2 (Ago-2), Dcr-1 and Ago-1, also resulted in a significant increase in replication of the two DENV strains tested, and the magnitude of this increase was similar to that resulting from Dcr-2 knockdown.These findings indicate that DENV can replicate in Drosophila S2 cells and that the RNAi pathway plays a role in modulating DENV replication in these cells. S2 cells offer a useful cell culture model for evaluation of the interaction between DENV and the RNAi response.

Dataset Information

Analysis of replication intermediates indicates that Drosophila melanogaster mitochondrial DNA replicates by a strand-coupled theta mechanism.

Publications

Analysis of replication intermediates indicates that Drosophila melanogaster mitochondrial DNA replicates by a strand-coupled theta mechanism.

Similar Datasets

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