Loss of autophagy causes a synthetic lethal deficiency in DNA repair

Abstract

(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the errorfree DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.autophagy | DNA repair | cell death | synthetic lethality T he preservation of genome integrity is critical for the prevention of human disease. In addition, the maintenance of proteome integrity is also considered central to healthy cellular homeostasis. Macroautophagy, hereafter referred to as autophagy, is a process that is paramount in counteracting damage to cytoplasmic constituents (1). Upon initiation of autophagy, double-membraned vesicles termed "autophagosomes" form to encapsulate cargoes including damaged or misfolded proteins and organelles. These vesicles ultimately fuse with lysosomes and the acidic hydrolases provided by the lysosome degrade cargoes into constituent parts, which can be recycled into biosynthetic pathways or in some situations, further catabolized to produce energy for the cell (1). Autophagy functions at basal levels in virtually all cells and is a major mechanism for protein turnover and the only known mechanism for degradation of organelles (1). Due to its crucial role in maintaining cytoplasmic and therefore cellular homeostasis, perturbations in autophagy have been reported to be an important contributing factor in a spectrum of diseases, including Crohn's disease, lysosomal storage disorders, neurodegenerative diseases, and cancer (2-6).Autophagy operates in the cytoplasm and yet studies have shown that autophagy-deficient cells accumulate DNA damage (5). The reasons behind this observation, however, are not completely clear. Because the cellular environment of autophagy-deficient cells will cause accrual of damaged proteins with abnormal function and as a result accumulation of reactive oxygen species, it is easily conceivable that this will ultimately lead to a higher incidence of genetic lesions. However, even when autophagy is competent, our cells are already subject to an extremely high frequency of spontaneous DNA damage. The fact that this damage does not persist is ...

Autophagy Regulates Chromatin Ubiquitination in DNA Damage Response through Elimination of SQSTM1/p62

“…Therefore, we monitored HR using the direct-repeat (DR)-GFP reporter and NHEJ using the pEJ5-GFP reporter. As shown in Figures 6A, 6B, S5A, and S5B, consistent with a previous report (Liu et al, 2015), autophagy inhibition reduced the efficiency of HR, but not NHEJ. More importantly, the reduction could be rescued by p62 knockdown (Figures 6A, 6B, S5A, and S5B), indicating that autophagy impairs DNA repair in a p62-dependent manner.…”

“…In our study, we found that p62 regulates both NHEJ and HR, although autophagy inhibitor only suppresses HR ( Figures 6A and 6B). It was recently reported that loss of autophagy leads to a defect in DSB repair by HR by reducing Chk1 abundance and phosphorylation (Liu et al, 2015). However, in our system, p62 did not affect Chk1 status, suggesting that p62-impaired DNA repair may rely on another pathway (for example, RNF168) ( Figures S5C-S5E).…”

“…In addition to controlling ROS production, persistence of DNA damage in autophagydeficient cells may be critically driven by a defect in DNA repair. Loss of autophagy critically impairs homologous recombination (HR), an important DNA repair mechanism, resulting from enhanced degradation of checkpoint kinase 1 (Chk1) (Liu et al, 2015). In addition, autophagy-mediated HP1 degradation is required for RAD6 induced HR-directed DNA DSB repair and subsequent changes in chromatin structure (Chen et al, 2015).…”

Autophagy regulates DNA repair by modulating histone ubiquitination

“…The two major pathways for repair of DSBs are homologous recombination (HR) and nonhomologous end joining (NHEJ). Consistent with a previous report, 8 autophagy inhibition reduced the efficiency of HR but not NHEJ. More importantly, this reduction could be rescued by p62 knockdown, indicating that autophagy impairs DNA repair in a p62-dependent manner.…”

Retrograde signaling from autophagy modulates stress responses

“…Enforced p62 accumulation induces ROS production, DNA damage, aneuploidy, and tumorigenesis (84). Furthermore, inhibition of autophagy leads to increased proteasomal activity and results in enhanced degradation and reduced abundance of Chk1, an essential factor for homologous recombination, and to a corresponding deficit in DNA repair of double-strand breaks (85).…”