7920 Nucleic Acids Research, 2012, Vol. 40, No. 16 Poisson distribution of lesions and a similar mtDNA copy number per cell, even at 50 and 100 J/m2 35 and 20%, respectively, of mtDNA genomes would be predicted to remain undamaged. Thus, in principle, the reduction in mtDNA damage observed here could result from dilution of damage rather than removal; however, mtDNA copy number did not increase during the recovery period at any UV dose as measured by real-time PCR (Figure 1c). In fact, there was a signiﬁcant decrease in copy number across treatments by 48 h post exposure (including controls; Figure 1d); this decrease was not altered by the level of damage (P = 0.3246 for treat- ment time interaction). Therefore, the decrease in mtDNA damage levels cannot be explained by dilution alone, and must, at least in part, be a result of removal of damaged genomes. Mitochondrial dynamics and autophagy are involved in removal of persistent mtDNA damage We next tested whether mitochondrial fusion, ﬁssion and autophagy assist in removal of mtDNA damage. Fusion, ﬁssion and autophagy genes were knocked down using RNAi in adult glp-1 C. elegans. Adult C. elegans were then dosed with 50 J/m2 and nuclear and mtDNA damage was measured immediately and 120 h post exposure. These RNAi experiments were carried out at 15 C except for a 15-h sterilization period at 25 C because we observed higher RNAi efﬁciency in our RNAi-positive control at the lower incubation tempera- ture. We extended the DNA damage removal period from 72 to 120 h because the lower incubation temperature slowed removal. We considered examining removal at later time points, but chose not to based on our previous observation that mtDNA copy number declines with age (52,67), as does mitochondrial function in general (70), effects that could confound our measurements. Although the damage removal observed during this 120 100 s n iso 80 e l AN 60 D t m 40 % 20 0 * L4440 eat-3 fzo-1 drp-1 atg-18 bec-1 unc-51 pink-1 pdr-1 Figure 2. Fusion, ﬁssion and autophagy gene knockdown inhibits mtDNA damage removal. mtDNA lesions are removed in empty vector control (L4440) by 30–40% 120 h post a single UVC exposure in post-mitotic adult C. elegans (glp-1). RNAi knockdown of eat-3, fzo-1, drp-1, bec-1, unc-51 and pink-1 inhibited mtDNA damage removal (inhibition of removal was determined by a lack of a signiﬁcant difference between 0 and 120 h lesion frequency within each RNAi treatment). Two-way ANOVA indicated a signiﬁcant interaction between RNAi and recovery (P < 0.05). Asterisks denote a signiﬁcant difference between 0 h and 120 h mtDNA lesions within RNAi treatment (Fisher’s PLSD, P < 0.05). Percent mtDNA lesions remaining after 120 h was calculated based on 0 h lesion frequency within each RNAi treatment. Bars ± SEM. timecourse was not complete, it was sufﬁcient for analysis and replicable in multiple experiments. In our empty vector control (L4440), there was 30% removal of mtDNA damage 120 h after a single UVC exposure (Figure 2). RNAi knockdown of fusion genes fzo-1 and eat-3, ﬁssion gene drp-1, and autophagy/ mitophagy genes bec-1, unc-51 and pink-1 abolished detectable removal of UVC-induced mtDNA damage. No increase in mtDNA copy number was observed in any sample as measured by real-time QPCR (Supplementary Figure S1), and decreases were observed in some cases. Repair of UVC-induced nDNA damage was not affected by RNAi (Supplementary Figure S2). These data demonstrate that fusion, ﬁssion and autophagy are required for removal of UVC-induced mtDNA damage. Persistent mtDNA damage induces autophagy without detectable changes in mitochondrial morphology Given the requirement for mitochondrial dynamics and autophagy genes in removal of mtDNA damage, we evaluated the effect of persistent mtDNA damage on mitochondrial morphology and autophagy following UVC exposure in adult C. elegans. To assess mitochon- drial morphology, young adult wild-type nematodes con- taining an extrachromosomal Pmyo-3::matrixGFP, which express GFP in the mitochondrial matrix of muscle cells (62), were exposed to a single dose of 50 J/m2 and mitochondrial morphology was assessed via confocal ﬂuorescence microscopy at 24 and 48 h post exposure. Mitochondrial morphology was quantiﬁed using form factor as a measure of mitochondrial elongation and mean area/perimeter ratio as a measure of mitochondrial interconnectivity (63,64). As shown in Figure 3a–b, no clear differences were observed in mitochondrial elong- ation or interconnectivity between control and UVC exposed nematodes at any time point. We observed that within individual nematodes and across treatments there 0 h 120 h * RNAi *