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Updated: Jul 24, 2026
Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
Published on: September 11, 2022
In vivo homologous recombination intermediates of yeast mitochondrial DNA analyzed by electron microscopy
Abstract:
To study the structure of in vivo mitochondrial DNA recombination intermediates in Saccharomyces cerevisiae, we used a deletion mutant of the wild type mitochondrial genome. The mtDNA of this petite is composed of a direct tandem repetition of an approximately 4,600 bp monomer repeat unit with a unique HhaI restriction enzyme site per repeat. The structure of native mtDNA isolated from log phase cells, and mtDNA crosslinked in vivo with trioxsalen plus UVA irradiation, was studied by electron microscopy. Both populations contained crossed strand "Holliday" type recombination intermediates. Digestion of both non-crosslinked and crosslinked mtDNA with the enzyme HhaI released X and H shaped structures composed of two monomers. Electron microscopic analysis revealed that these structures had pairs of equal length arms as required for homologous recombination intermediates and that junctions could occur at points along the entire monomer length. The percentage of recombining monomers in both non-crosslinked and trioxsalen crosslinked mtDNA was calculated by quantitative analysis of all the structures present in an HhaI digest. The relationship between these values and the apparent dispersive replication of mtDNA in density-shift experiments and mtDNA fragility during isolation is discussed.
Insights
This study visualizes mitochondrial DNA recombination in yeast, identifying Holliday junctions in vivo. These findings offer insights into mitochondrial genome stability and repair mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Mitochondrial DNA (mtDNA) recombination is crucial for maintaining mitochondrial genome integrity.
- Understanding recombination intermediates in vivo is essential for studying mtDNA stability and repair.
- Previous studies have lacked direct visualization of recombination structures within the cell.
Purpose of the Study:
- To investigate the in vivo structure of mitochondrial DNA recombination intermediates.
- To characterize the nature of these intermediates using a specific yeast deletion mutant.
- To correlate structural findings with mtDNA replication and isolation procedures.
Main Methods:
- Utilized a Saccharomyces cerevisiae deletion mutant with a tandemly repeated mtDNA monomer.
- Employed electron microscopy to examine native and in vivo crosslinked mtDNA.
- Used HhaI restriction enzyme digestion to release specific recombination structures.
Main Results:
- Identified "Holliday" type crossed strand recombination intermediates in both native and crosslinked mtDNA.
- Observed X and H shaped structures composed of two monomers after HhaI digestion.
- Demonstrated that recombination junctions can occur along the entire monomer length, with equal arm lengths.
Conclusions:
- Directly visualized in vivo mitochondrial DNA recombination intermediates, confirming their Holliday junction structure.
- Provided evidence for homologous recombination occurring throughout the mitochondrial genome.
- Discussed the implications of these findings for mtDNA replication, stability, and potential fragility during isolation.
