7924 Nucleic Acids Research, 2012, Vol. 40, No. 16 shown); however, 3-MA exposure did exacerbate UVC-induced L3 arrest (Figure 7). It is interesting that knockout of fusion resulted in complete larval arrest with no recovery, whereas knockout of autophagy led to a more moderate increase in larval arrest with delayed recovery. If removal of UVC-induced mtDNA damage were the sole driver of recovery then knockout of these two pathways would lead to similar results. Mitochondrial fusion has been shown to be critical for mtDNA integrity and recovery from mitochondrial dysfunction in part because it aids in mtDNA replication, which is critical for recovery from UVC-induced larval arrest as shown earlier in the text and in functional complementation (80) that results from mixing damaged mtDNA among undamaged mtDNA. These results suggest that removal of damaged mtDNA does aid in recovery from mitochondrial dysfunc- tion; however, mtDNA replication and mitochondrial fusion are necessary for recovery. We suggest that enhanced mitochondrial fusion as a result of drp-1 or pink-1 knockout may explain why knockout of these genes did not affect larval arrest or recovery although both of these are involved in mtDNA damage removal. As previously mentioned, measuring removal of mtDNA damage in proliferating cells is confounded by dilution of damage associated with cell replication. C. elegans larval development is marked by a 2-fold increase in somatic cells, a 5-fold increase in mtDNA copy number and development of approximately 250 germ cell nuclei (52). Therefore, we were not able to directly test if mutations in these genes prevented removal of damage or if arrested nematodes had more persistent mtDNA damage compared with non-arrested nematodes because the developmental changes would have confounded those measurements. Nonetheless, our genetic and pharmacologic data allow us to conclude that mitochondrial fusion and autophagy play an import- ant role in recovery from UVC-induced mitochondrial dysfunction. DISCUSSION Mitochondrial dynamics and autophagy play critical roles in response to persistent mitochondrial DNA damage MtDNA integrity is critical to mitochondrial function and is more vulnerable than nDNA to common types of environmentally induced damage (6–11). This vulnerabil- ity is exacerbated by the fact that mitochondria lack NER, the pathway required to repair helix distorting and bulky DNA damage induced by important environmental genotoxins including UVC, mycotoxins and PAHs (12–15). Thus, at least in most species (ﬁssion yeast is an exception (81)), mtDNA damage induced by such agents is irreparable. Our experiments indicate that mitochondrial dynamics and autophagy protect against persistent mtDNA damage; we suggest that this results both from a role in removal of damaged mtDNAs and from a role in protecting mitochondrial function in the face of mtDNA damage. Figure 8. Proposed model of the effect of mitochondrial fusion, ﬁssion and autophagy processes on persistent mtDNA damage-induced mito- chondrial dysfunction and removal of mtDNA damage. Persistent mtDNA damage leads to mitochondrial dysfunction (e.g. reduced ATP levels and larval arrest). Mitochondrial fusion protects against dysfunction, as does autophagy, presumably by promoting functional complementation, mtDNA replication and removal of dysfunctional mitochondria (red solid lines). Mitochondrial ﬁssion and autophagy promote mtDNA damage removal (green dashed lines), whereas mito- chondrial fusion is indirectly required for mtDNA damage removal because it is required to preserve basic mitochondrial function and mtDNA replication (dashed green line denotes the indirect effect of fusion on mtDNA damage removal). We propose a model (Figure 8) whereby persistent mtDNA damage leads to mitochondrial dysfunction. Mitochondrial fusion protects against dysfunction, as does autophagy, presumably by promoting functional complementation, mtDNA replication and removal of dysfunctional mitochondria (these processes are further discussed below). Mitochondrial genomes carrying persist- ent damage can be removed by mitochondrial ﬁssion and subsequent autophagy of dysfunctional daughter mitochondria, which is observed in our experiments as a mitochondrial ﬁssion- and autophagy-dependent removal of mtDNA damage. It is not intuitive that mitochondrial fusion would be required for the removal of damaged mtDNAs, but we propose that fusion is so critical for mitochondrial function (as supported by our larval arrest results) and mitochondrial biogenesis that the lack of fusion results in severe mitochondrial dysfunction that incapacitate the processes required for removal. Although additional experiments will be required to fully test this model, it is consistent with our data and supported by a signiﬁcant body of literature, as discussed later in the text. Mitochondrial fusion, ﬁssion and autophagy are required for removal of persistent mtDNA damage Irreparable, helix-distorting mtDNA damage is persistent but our results indicate that this damage is removed slowly in vivo with signiﬁcant removal observed by 72 h post exposure. These data are consistent with other literature indicating the persistence of UV-induced photodimers up to 48 h (15,17,18,66,67), although one study reported a 40% reduction in UVC-induced photodimers in mtDNA of mouse cells by 24 h (82). We considered the role of mitochondrial dynamics and degradation via autophagy in this removal. Autophagy is the primary mechanism of removal for mitochondria and can be both non-selective, such as during starvation (31), or selective meaning that isolated, dysfunctional mitochondria with low MP are