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Replication of mitochondrial DNA in mouse L cells and their thymidine kinase - derivatives: displacement replication
Abstract:
Thymidine kinase(-) lines of mouse L cells incorporate thymidine exclusively into mitochondrial DNA. This fact permits assessment of labeled mitochondrial DNA components in ethidium bromide-cesium chloride gradients. Contaminating nuclear DNA is unlabeled and need not be removed. Elimination of the DNase treatment of purified mitochondria reveals that the replicative forms that exhibit displacement replication up to full genome size are covalently-closed circular molecules. Denaturation followed by brief renaturation of these larger replicating molecules produces closed-circular DNA with a deficiency of Watson-Crick turns, appearing as single-strand loops. This result suggests that displacement replication proceeds with nicking and rapid closure of the covalently-closed circular template.
Insights
Mouse L cells lacking thymidine kinase incorporate thymidine into mitochondrial DNA, allowing study of its replication. This reveals mitochondrial DNA replication involves nicking and rapid closure of circular DNA templates.
Area of Science:
- Mitochondrial DNA replication
- Molecular biology
- Cellular genetics
Background:
- Thymidine kinase-deficient (TK-) mouse L cells exclusively incorporate thymidine into mitochondrial DNA.
- This characteristic enables the study of mitochondrial DNA synthesis without nuclear DNA contamination.
Purpose of the Study:
- To investigate the molecular mechanisms of mitochondrial DNA replication.
- To characterize the structure of replicating mitochondrial DNA molecules.
Main Methods:
- Utilizing TK- mouse L cells for selective thymidine incorporation.
- Employing ethidium bromide-cesium chloride density gradients for DNA separation.
- Analyzing purified mitochondria, with and without DNase treatment, to study DNA forms.
Main Results:
- Replicative forms of mitochondrial DNA exist as covalently-closed circular molecules.
- These molecules exhibit displacement replication up to full genome size.
- Denaturation and renaturation of replicating molecules reveal single-strand loops, indicating template nicking and closure.
Conclusions:
- Mitochondrial DNA replication proceeds via a displacement mechanism.
- The process involves nicking and rapid re-ligation of the covalently-closed circular template DNA.