In contrast, another mouse strain overexpressing TFAM has increased steady-state levels of TWINKLE.
Western blot analysis to assess the steady-state levels of TWINKLE protein showed the absence of the TWINKLE protein in knockout hearts from an early age (Fig. 1E).
Given the observations that Twinkle knockout leads to mtDNA depletion and that TWINKLE overexpression leads to an increased mtDNA copy number, we investigated whether there is a general connection between the levels of TWINKLE protein and mtDNA.
Interestingly, expression levels of Twinkle are very similar to expression levels of DNA Pol gamma I, a dual-targeted DNA polymerase that has been shown to play a role in plant organelle DNA replication and repair [ 24].
Mice with general TFAM overexpression showed increased steady-state levels of TWINKLE in all analyzed tissues, consistent with the idea that the mtDNA copy number increase is linked to increased mtDNA replication (Fig. 6C).
Low expression levels of Twinkle K319E lead to the appearance of a sharp double-stranded bubble-arc in a fragment containing the non-coding region of mtDNA, while the club-headed double bubble-arc typical for RNA-rich initiation intermediates was reduced.
The results show relatively high levels of the Twinkle protein in weeks 1 3 of growth, with a subsequent rapid drop in levels until the protein is nearly undetectable after week 5.
The similar levels of expression of Twinkle and the organelle-localized DNA polymerases [ 25] suggest that Twinkle may play a role in replication of organelle DNA.
However, heart-specific Tfam knockout mice also showed upregulation of TWINKLE levels, suggesting that mtDNA replication is activated as a futile compensatory response to counteract the profound mtDNA depletion (Fig. 6C).
