Autophagy in DNA Damage Response

DNA damage response (DDR) involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1). mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADP)ribose polymerase 1 (PARP-1), Mre11–Rad50–Nbs1 (MRN) complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy

DNA damage induced during mitosis undergoes DNA repair synthesis.

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase

Quantitative mechanisms of DNA damage sensing and signaling

DNA damage occurs abundantly during normal cellular proliferation. This necessitates that cellular DNA damage response and checkpoint pathways monitor the cellular DNA damage load and that DNA damage signaling is quantitative. Yet, how DNA lesions are counted and converted into a quantitative response remains poorly understood. We have recently obtained insights into this question investigating DNA damage signaling elicited by single-stranded DNA (ssDNA). Intriguingly, our findings suggest that local and global DNA damage signaling react differentially to increasing amounts of DNA damage. In this mini-review, we will discuss these findings and put them into perspective of current knowledge on the DNA damage response

Radiation induced DNA damage responses

The amazing feature of ionising radiation (IR) as a DNA damaging agent is the range of lesions it induces. Such lesions include base damage, single strand breaks (SSBs), double strand breaks (DSBs) of varying complexity and DNA cross links. A range of DNA damage response mechanisms operate to help maintain genomic stability in the face of such damage. Such mechanisms include pathways of DNA repair and signal transduction mechanisms. Increasing evidence suggests that these pathways operate co-operatively. In addition, the relative impact of one mechanism over another most probably depends upon the cell cycle phase and tissue type. Here, the distinct damage response pathways are reviewed and the current understanding of the interplay between them is considered. Since DNA DSBs are the major lethal lesion induced by IR, the focus lies in the mechanisms responding to direct or indirectly induced DSBs

TDP1 deficiency sensitizes human cells to base damage via distinct topoisomerase I and PARP mechanisms with potential applications for cancer therapy

Base damage and topoisomerase I (Top1)-linked DNA breaks are abundant forms of endogenous DNA breakage, contributing to hereditary ataxia and underlying the cytotoxicity of a wide range of anti-cancer agents. Despite their frequency, the overlapping mechanisms that repair these forms of DNA breakage are largely unknown. Here, we report that depletion of Tyrosyl DNA phosphodiesterase 1 (TDP1) sensitizes human cells to alkylation damage and the additional depletion of apurinic/apyrimidinic endonuclease I (APE1) confers hypersensitivity above that observed for TDP1 or APE1 depletion alone. Quantification of DNA breaks and clonogenic survival assays confirm a role for TDP1 in response to base damage, independently of APE1. The hypersensitivity to alkylation damage is partly restored by depletion of Top1, illustrating that alkylating agents can trigger cytotoxic Top1-breaks. Although inhibition of PARP activity does not sensitize TDP1-deficient cells to Top1 poisons, it confers increased sensitivity to alkylation damage, highlighting partially overlapping roles for PARP and TDP1 in response to genotoxic challenge. Finally, we demonstrate that cancer cells in which TDP1 is inherently deficient are hypersensitive to alkylation damage and that TDP1 depletion sensitizes glioblastoma-resistant cancer cells to the alkylating agent temozolomide

Inflammation-induced DNA damage and damage-induced inflammation: a vicious cycle

Inflammation is the ultimate response to the constant challenges of the immune system by microbes, irritants or injury. The inflammatory cascade initiates with the recognition of microorganism-derived pathogen associated molecular patterns (PAMPs) and host cell-derived damage associated molecular patterns (DAMPs) by the pattern recognition receptors (PRRs). DNA as a molecular PAMP or DAMP is sensed directly or via specific binding proteins to instigate pro-inflammatory response. Some of these DNA binding proteins also participate in canonical DNA repair pathways and recognise damaged DNA to initiate DNA damage response. In this review we aim to capture the essence of the complex interplay between DNA damage response and the pro-inflammatory signalling through representative examples

The DNA damage response acts as a safeguardagainst harmful DNA–RNA hybrids ofdifferent origins

Despite playing physiological roles in specific situations, DNA–RNA hybrids threat genome integrity. To investigate how cells do counteract spontaneous DNA–RNA hybrids, here we screen an siRNA library covering 240 human DNA damage response (DDR) genes and select siRNAs causing DNA–RNA hybrid accumulation and a significant increase in hybrid‐dependent DNA breakage. We identify post‐replicative repair and DNA damage checkpoint factors, including those of the ATM/CHK2 and ATR/CHK1 pathways. Thus, spontaneous DNA–RNA hybrids are likely a major source of replication stress, but they can also accumulate and menace genome integrity as a consequence of unrepaired DSBs and post‐replicative ssDNA gaps in normal cells. We show that DNA–RNA hybrid accumulation correlates with increased DNA damage and chromatin compaction marks. Our results suggest that different mechanisms can lead to DNA–RNA hybrids with distinct consequences for replication and DNA dynamics at each cell cycle stage and support the conclusion that DNA–RNA hybrids are a common source of spontaneous DNA damage that remains unsolved under a deficient DDR.European Research Council (ERC2014AdG669898TARLOOP)Worldwide Cancer Research (WCR15-00098

Requirement of the FATC domain of protein kinase Tel1 for localization to DNA ends and target protein recognition

Two large phosphatidylinositol 3-kinase-related protein kinases (PIKKs), ATM and ATR, play a central role in the DNA damage response pathway. PIKKs contain a highly conserved extreme C-terminus called the FRAP-ATM-TRRAP-C-terminal (FATC) domain. In budding yeast, ATM and ATR correspond to Tel1 and Mec1, respectively. In this study, we characterized functions of the FATC domain of Tel1 by introducing substitution or truncation mutations. One substitution mutation, termed tel1-21, and a truncation mutation, called tel1- Î"C, did not significantly affect the expression level. The tel1-21 mutation impaired the cellular response to DNA damage and conferred moderate telomere maintenance defect. In contrast, the tel1-Î"C mutation behaved like a null mutation, conferring defects in both DNA damage response and telomere maintenance. Tel1-21 protein localized to DNA ends as effectively as wild-type Tel1 protein, whereas Tel1-Î"C protein failed. Introduction of a hyperactive TEL1-hy mutation suppressed the tel1-21 mutation but not the tel1-Î"C mutation. In vitro analyses revealed that both Tel1-21 and Tel1-Î"C proteins undergo efficient autophosphorylation but exhibit decreased kinase activities toward the exogenous substrate protein, Rad53. Our results show that the FATC domain of Tel1 mediates localization to DNA ends and contributes to phosphorylation of target proteins. © 2015 Ogi, Goto, Ghosh, et al