1. Twinkle Mutants
K319E sample shows decreased mtDNA copy numbers after 24 hours dox treatment
Observed in combing experiments and Antoine’s qPCR data
Dosed with:
Dox for 14 hours
CldU for 15 min
IdU for 60 min
media for 90 min

2. MH-6
K319E-3
In process of quantitating the number of labeled molecules compared to the yoyo images

3. MH6 - no dox

4. MH hour dox

5. K319E-3 No Dox

6. K319E Hour Dox

7. Twinkle Statistics MH6 No dox MH6 14 hr dox K319E3 No dox
Group 1 – 35 (61%)
Group 2 – 22 (39%)
MH6 14 hr dox
Group 1 – 92 (75%)
Group 2 – 30 (25%)
K319E3 No dox
Group 1 – 51 (61%)
Group 2 – 32 (39%)
K319E3 14 hr dox
Group 1 – 36 (71%)
Group 2 – 15 (29%)

8. Overexpressing TWINKLE Altered the Replication Profile
Group 1
CldU
IdU
Group 2
We have observed using our single molecule assay, that the overexpression of this protein induces its own phenotype in both the WT and mutant cell lines, not previously observed in less sensitive methods of interrogation. Therefore, we need a cleaner genetic system to investigate our hypothesis, so we are using CRISPRs to create inducible knockouts of endogenous Twinkle
IdU
CldU
IdU