‘mitophagy’ (32) is responsible for clearance of paternal mtDNA in sperm of Caenorhabditis elegans (33,34), of mitochondria during erythrocyte maturation (35) and has emerged as a more speciﬁc degradation mechanism for dysfunctional mitochondria in somatic cells (30,32,36–38). Although the detailed mechanism of dys- functional mitochondrial clearance by mitophagy has yet to be resolved, current research suggests that spontaneous or stress-induced depolarization results in isolation of dysfunctional mitochondria followed by removal by autophagy (30,39–43). The Parkinson’s disease-associated genes encoding Parkin and PINK1 have been shown to selectively accumulate on mitochondria with reduced MP and facilitate mitophagy under certain conditions (44–47). The role of mitophagy in removal of dysfunctional mitochondria with damaged DNA has not been previously addressed but this could play an important role in preventing ﬁxation of mtDNA mutations and mitochondrial-mediated apoptosis or necrosis by removal of unstable mitochondria (48,49). The goals of this research were to determine the long-term fate of helix-distorting mtDNA damage and investigate the role of autophagy and mitochondrial dynamics in removal of and recovery from this mtDNA damage. Using UVC, we induced helix-distorting mtDNA lesions in adult C. elegans with and without RNAi knockdown of autophagy, fusion and ﬁssion genes and measured mtDNA damage over time using quantitative polymerase chain reaction (qPCR). We show that UVC-induced mtDNA lesions are removed slowly over 72 h in adult C. elegans and that this removal is dependent upon genes involved in autophagy and mitochondrial dynamics. To better understand the implications of per- sistent mtDNA damage removal in vivo, we investigated in C. elegans the effect of UVC-induced mtDNA damage on the frequency and duration of larval arrest, a well-described phenotype mediated by mitochondrial function that serves as an indicator of overall mitochon- drial health (50–53). We show that high levels of persistent mtDNA damage induce L3 larval arrest and mitochon- drial dysfunction in C. elegans. Furthermore, recovery from larval arrest is dependent on mitochondrial fusion, mtDNA replication and removal of damaged mtDNA via autophagy. Thus, we show for the ﬁrst time that, although persistent, UVC-induced mtDNA lesions are removed gradually and that mitochondrial dynamics and autophagy are involved in removal of and recovery from persistent mtDNA damage. MATERIALS AND METHODS Caenorhabditis elegans strains and culture Populations of C. elegans were maintained on K agar plates seeded with OP50 bacteria unless otherwise stated. N2 (wild-type), JK1107 glp-1(q224), VC1024 pdr-1(gk448), CB369 unc-51(e369) were obtained from the Caenorhabditis Genetics Center (University of Minnesota). DA631 eat-3(ad426), BC10210 fzo-1(tm1133) and N2+Ex[myo-3::mls::matrixGFP+ rol-6] were provided by Alexander van der Bliek, Nucleic Acids Research, 2012, Vol. 40, No. 16 7917 University of California (Los Angeles, CA, USA). drp-1 (tm1108) was provided by Ding Xue, University of Colorado (Boulder, CO, USA). Guy Caldwell, University of Alabama (Tuscaloosa, AL, USA), provided UA86 pink-1(tm1779); Pdat-1::GFP; Pdat-1:: a-syn [baIn11]. Luminescent PE255 (feIs5) were provided by Christina Lagido, University of Aberdeen (Aberdeen, UK). Alicia Melendez, Queens College, City University of New York (Flushing, NY, USA) kindly provided QU1 izEx1[Plgg-1::gfp::lgg-1+rol-6]. Quantitative PCR DNA damage analysis was performed using qPCR optimized for whole C. elegans as described in Boyd et al. (54) with some modiﬁcations. glp-1 adults were picked at a 1 worm to 15 ml lysis buffer ratio. In RNAi experiments, nuclear and mtDNA copy number for each QPCR sample determined by quantitative, real-time PCR was used to normalize for copy number variation between samples. This assay relies on the ability of DNA damage to block progression of the DNA polymerase used for PCR; thus, the amount of ampliﬁcation is inversely related to the amount of DNA damage (55,56). In each sample, a long ( 10 kb) nuclear and mitochondrial genomic region is ampliﬁed. The long product is used to calculate lesion frequency as compared with control samples. DNA damage and repair quantiﬁcation Synchronized glp-1 L1 larvae were obtained by bleach- sodium hydroxide isolation of eggs and liquid egg hatch in K+ medium (54). Nematodes were grown to young adult stage at 25 C, then transferred to K-agar plates without OP50 and exposed to 50 J/m2 UVC using an ultra- violet lamp (UVLMS-38 EL Series 3UV Lamp, UVP, Upland, CA, USA) with peak emission at 254 nm. Doses were quantiﬁed using a UVX digital radiometer. Nematodes were lysed at 0, 24, 48 and 72 h after exposure. Nematodes were incubated at 25 C during recovery periods. Analysis was performed on 7–12 sample tubes (6 nematodes/tube) for each treatment con- dition generated from at least two independent, time-separated experiments creating an ‘n’ of 7–12. At least two PCR technical replicates were carried out for each sample. Lesion frequency in UVC-treated samples was calculated relative to RNAi clone untreated controls. Global analysis of variance (ANOVA) analysis was used to determine effects and interactions of treat- ment, recovery and RNAi clone (Statview 5.0.1). When warranted by global ANOVA, Fisher’s PLSD was used to determine the effect of individual variables. Mitochondrial copy number analysis Absolute mitochondrial copy number was measured by quantitative, real-time PCR as described in Bratic et al. (57) using the lysed worm samples collected for QPCR as described earlier in the text. For each sample, three technical replicates were averaged from a single real-time PCR run.