the third UVC exposure using a Mitocell (MT200) respir- ation chamber with magnetic stirrer and 1302 Clark-type microcathode oxygen electrode attached to a 782 oxygen meter (Strathkelvin Instruments, Glasgow, UK) as described in (65). At 0 and 24 h, 1000 nematodes were sorted into four 1.5 ml microcentrifuge tubes using a COPAS Biosort (Union Biometrica, Holliston, MA, USA) with the same gate used for untreated and treated samples. At 48 h, 500 nematodes were sorted. The UVC- exposed group was sorted into two populations: large (non-arrested) and small (arrested). The large or non-arrested gate was deﬁned using the control popula- tion that is at L4 or older. The small or arrested gate was deﬁned as any nematode smaller than the control popula- tion. Immediately before measurement worms were centrifuged, 100 ml of pelleted worms were added to the Mitocell chamber and oxygen consumption was measured for 4 min once linear. The rate of oxygen consumption was calculated within the linear range for each sample and two-way ANOVA followed by Fisher’s PLSD (if applic- able) were used to identify signiﬁcant effects of treatment and recovery (Statview 5.0.1). RESULTS UVC-induced mtDNA damage is removed over time Studies in cell culture and C. elegans have established the persistence of mtDNA damage up to 48 h post exposure to Nucleic Acids Research, 2012, Vol. 40, No. 16 7919 UVC (17,66,67) but further time points have not been investigated, partially because of the confounding effect of dilution of mtDNA damage encountered with proliferating cells in culture. We utilized the germ-line pro- liferation defective mutant strain glp-1(q224) to investigate the persistence of UVC-induced mtDNA damage. Adult glp-1 raised at 25 C have post-mitotic somatic cells and few (6–8) germ cells (68), such that changes in DNA damage can be attributed to repair or removal rather than dilution following cell replication (67). Radiation (UVC) of 254 nm serves as an excellent model for bulky adducts because it similarly induces helix-distorting lesions and it is primarily absorbed by nucleic acids, with little damage to other macromolecules; thus we utilized UVC to induce helix-distorting mtDNA lesions throughout our studies. Post-mitotic young adult glp-1 nematodes raised at 25 C were exposed to 0–100 J/m2 UVC and both nuclear and mtDNA damage was analyzed at 0, 24, 48 and 72 h post exposure via a highly sensitive QPCR assay that does not require differential extraction of nuclear and mtDNA (55,56,69). Over 72 h, 30–40% removal of mtDNA damage was observed at 50 and 100 J/m2 (Figure 1a). nDNA damage was repaired to control level by 72 h (Figure 1b). mtDNA replication occurs independently of nDNA replication, and while UV-induced lesions are likely to block replication (26), it is unlikely that all of the mitochondrial genomes per cell were damaged even at our highest doses. Assuming a Figure 1. mtDNA lesion frequency slowly decreases after a single dose of UVC in post-mitotic adult C. elegans (glp-1). Nematodes were exposed to UVC and analyzed for DNA damage immediately (0 h), or after 24, 48 or 72 h via QPCR. (a) mtDNA damage is removed by 40% over 72 h at 50 and 100 J/m2. Two-way ANOVA indicated a signiﬁcant effect of recovery (P < 0.0001) and a recovery treatment interaction (P < 0.05). Asterisks denote a signiﬁcant difference compared with 0 h lesion frequency within each UVC treatment (Fisher’s PLSD, P < 0.05). (b) nDNA damage is repaired following a single dose of UVC. Two-way ANOVA indicated a signiﬁcant effect of treatment (P < 0.0001) and a recovery treatment interaction (P < 0.0001). Asterisks denote a signiﬁcant difference compared with undosed control lesion frequency at each recovery timepoint (Fisher’s PLSD, P < 0.05). (c) No signiﬁcant increase in mtDNA copy number was observed during the recovery period. (d) mtDNA copy number signiﬁcantly decreased during the recovery period irrespective of UVC exposure. Two-way ANOVA indicated a signiﬁcant effect of time (P < 0.0001) but no signiﬁcant treatment effect (P = 0.4714) or treatment time interaction (P = 0.3246). Asterisks denote a signiﬁcant difference compared with 0 h mtDNA copy number (Fisher’s PLSD, P < 0.05). Bars ± SEM.