L3 arrest is inﬂuenced by lingering low (below our limit of detection) levels of nDNA damage. We note, however, that in a previous study that employed a single 10 J/m2 dose of UVC, which should result in a similar maximal level of nDNA damage to that which we measured throughout the larval arrest protocol ( 0.8 lesions/ 10 kb) but a much lower level of mtDNA damage (also 0.8 lesions/10 kb), no effect on larval development was observed (78). mtDNA replication is critical for recovery from persistent mtDNA damage At serial UVC doses <25 J/m2, nematodes recover from larval arrest over time (Figure 5a). This recovery may be facilitated by replacement of UVC-damaged mitochon- drial genomes. Ethidium bromide is a DNA intercalating agent that preferentially targets mtDNA over nDNA and blocks replication (79). Previous studies indicate that inhibition of mtDNA replication by ethidium bromide results in L3 arrest and depletion of mtDNA (52). If recovery from UVC-induced L3 arrest is aided by dilution of damaged mtDNA via replication of undam- aged genomes, then co-exposure to UVC and ethidium bromide should exacerbate larval arrest. We selected an ethidium bromide concentration (5 mg/ml) that results in a similar frequency and duration of larval arrest as UVC 10 J/m2. Co-exposure with UVC (10 J/m2) and ethidium bromide (5 mg/ml) dramatically exacerbated L3 arrest across all time points compared with UVC or chemical alone (Figure 6). This demonstrates the import- ance of mtDNA replication in recovery of mitochondrial function resulting from persistent mtDNA damage. Recovery from UVC-induced mtDNA damage involves mitochondrial fusion and autophagy To better understand the role of removal of mtDNA damage in recovery from UVC-induced L3 arrest, we compared L3 arrest in wild-type (N2) C. elegans with strains carrying mutations in fusion, ﬁssion and autophagy genes. If removal of persistent mtDNA * * * * * * )120 l o r t on100 c l L3≤ icam80 tsaonedem itt/rscnoeh6400 a % de rpa20 m o (c 0 Nucleic Acids Research, 2012, Vol. 40, No. 16 7923 damage plays a signiﬁcant role in vivo in recovery from UVC-induced mitochondrial dysfunction then knockout of these genes involved in removal will increase the frequency and duration of larval arrest compared with wild-type. As shown in Figure 7, signiﬁcant exacerbation of L3 arrest was observed in the fusion mutants fzo-1 and eat-3 and the autophagy mutant unc-51. Mutations in ﬁssion gene drp-1 or mitophagy genes pink-1 and pdr-1 did not signiﬁcantly exacerbate L3 arrest at any time point. We attempted to test other autophagy mutants but were unsuccessful because of embryonic lethality in homozy- gotes [as in the case of bec-1 (ok961)] or an inability of the mutant strain to recover from the 48-h L1 starvation period [as in the case of atg-18 (gk378)]. Since we were limited in our ability to test genetically for a role of autophagy in recovery from persistent mtDNA damage because of mutant inviability, we chemically inhibited autophagy with 3-MA. N2 nematodes were exposed to 10 mM 3-MA following the third UVC exposure and placement on food. At this dose, 3-MA exposure alone did not result in signiﬁcant larval arrest (data not Figure 6. Co-exposure to UVC and ethidium bromide exacerbates L3 2 arrest. Co-exposure to UVC (10 J/m ) and ethidium bromide (5 mg/ml) further exacerbates L3 arrest compared with UVC (asterisks) or chemical (hash) exposure alone at every time point (Fisher’s PLSD, P < 0.05). Bars ± SEM. * * 48 h 72 h 96 h * * N2 eat-3 fzo-1 drp-1 unc-51 pink-1 pdr-1 3-MA UVC 10 J/m2 Figure 7. Mutations in fusion and autophagy genes exacerbate L3 arrest following serial UVC exposure. Mutations in fusion genes fzo-1 and eat-3 and autophagy gene unc-51, as well as inhibition of autophagy with 3-MA, exacerbated L3 arrest compared with wild-type at 72 and 96 h. Asterisks denote a signiﬁcantly different effect of treatment on mutant compared with wild-type strain (two-way ANOVA, P < 0.05). Bars ± SEM.