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  • In conclusion as illustrated in Fig


    In conclusion, as illustrated in Fig. 5G, in response to MNNG-induced DNA damage, PARP-1 appears to have dual roles in determining the cell fate in response to MNNG: PARP-1 activation is the cause of non-apoptotic cell death via ATP depletion and PARP-1 activation is able to elicit a self-protective mechanism by induction of autophagy via the AMPK-mTOR pathway. Our findings thus provide novel insights into the complex relationship among alkylating agent-induced DNA damage, autophagy and cell death.
    Introduction The genome is constantly hit by multiple sources of exogenous and endogenous damage that compromise its integrity. Eukaryotic cells respond to the presence of genome injuries by activating surveillance mechanisms referred to as DNA damage or DNA integrity checkpoints. The inability to properly react to DNA damage results in genome instability, which in mammalian systems is linked to tumor development [1], [2], [3]. In Saccharomyces cerevisiae, DNA damage is initially detected by the Mec1/Ddc2 (ATR/ATRIP) and the clamp-like Ddc1-Rad17-Mec3 (‘9-1-1’) complexes. These checkpoint sensors are independently recruited to the sites of damage and trigger the activation of the Rad53 and Chk1 effector kinases in hexokinase inhibition a process mediated by the Rad9 and Mrc1 adaptors. In turn, the effector kinases act on the corresponding targets to promote the different cellular responses to cope with the DNA damage, including hexokinase inhibition arrest, stabilization of replication forks and activation of DNA repair [4]. Eukaryotic cells are equipped with a broad range of specialized DNA repair pathways to confront and eliminate the great variety of genomic insults of different nature that can arise during different cell cycle stages, but lesions occurring during S phase that can stall replication forks are particularly threatening [5], [6]. Thus, in addition to the repair pathways to remove the lesions, cells possess tolerance mechanisms, such as translesion synthesis (TLS) and template switching, that allow replication to continue despite the presence of DNA damage [7]. These tolerance pathways are critical for survival in the face of DNA damage. TLS is mediated by specialized polymerases that, in contrast to replicative polymerases, are able to insert nucleotides opposite damaged templates, although at the cost of increasing the mutagenesis rate. Therefore, this tolerance pathway must be tightly controlled. In yeast, TLS is performed by the Polη polymerase (encoded by the RAD30 gene), and by the Polζ polymerase composed by the Rev3 (catalytic) and Rev7 (regulatory) subunits [8]. In addition, the Rev1 protein also plays a critical role in TLS. Although Rev1 possesses deoxycytidyl transferase activity, its main TLS function is structural and does not rely on the catalytic activity [9], [10]. In eukaryotes, DNA damage tolerance is exquisitely controlled by ubiquitylation of the DNA sliding clamp PCNA at the lysine 164 [11]. Thus, Rad6/Rad18-dependent monoubiquitylation of PCNA-K164 triggers TLS, whereas polyubiquitylation of PCNA-K164 through Ubc13/Mms2/Rad5 induces the template-switch error-free mode of damage bypass by sister-strand recombination [7], [12], [13], [14]. Genome injuries do not occur on the naked DNA, but rather in the context of the highly organized chromatin. Indeed, during the recent years significant advances have been made in understanding the contribution of chromatin modifications to several aspects of the DNA damage response, such as detection, signaling and repair of the damage [15], [16], [17], [18], [19], [20]. However, little is known about how chromatin structure may impinge on DNA damage tolerance, although a recent report has described a role for the INO80 remodeling complex in DNA damage tolerance through modulation of PCNA ubiquitylation [21]. Methylation of lysine 79 in histone H3 (hereafter H3K79-me) by the Dot1 methyltransferase is one of the various histone modifications involved in the cellular responses to DNA damage. Dot1 orchestrates several aspects of chromosome metabolism both in mitotic and meiotic cells, including transcriptional silencing [22], [23], [24], [25], activation of the meiotic recombination checkpoint and regulation of recombination partner choice in meiosis [26], repair of double-strand breaks by sister-chromatid recombination in mitotic cells [27], and repair of IR- and UV-induced lesions [28], [29], [30], [31]. In addition, Dot1 participates in the DNA damage checkpoint in vegetative yeast cells being required for Rad9-mediated activation of the Rad53 effector kinase, at least during the G1-S cell cycle transitions [32], [33]. Moreover, we have recently reported that Dot1 negatively regulates the Polζ/Rev1-dependent pathway of tolerance to alkylating DNA damage. Indeed, deletion of DOT1 results in increased Rev3-dependent mutagenesis [34]. Dot1 is conserved form yeast to human; importantly, altered function of human DOT1L is linked to leukemia development [35], [36], [37], [38], [39].