Direct Activation of ATM by Resveratrol under Oxidizing Conditions

Resveratrol has been widely reported to reduce cancer progression in model systems and to selectively induce cell death in transformed cell lines. Many enzymes have been reported to respond to resveratrol in mammalian cells, including the Ataxia-Telangiectasia Mutated (ATM) protein kinase that acts in DNA damage recognition, signaling, and repair. Here we investigate the responses of ATM to resveratrol exposure in normal and transformed human cell lines and find that ATM autophosphorylation and substrate phosphorylation is stimulated by resveratrol in a manner that is promoted by reactive oxygen species (ROS). We observe direct stimulatory effects of resveratrol on purified ATM in vitro and find that the catalytic efficiency of the kinase on a model substrate is increased by resveratrol. In the purified system we also observe a requirement for oxidation, as the effect of resveratrol on ATM signaling is substantially reduced by agents that prevent disulfide bond formation in ATM. These results demonstrate that resveratrol effects on ATM are direct, and suggest a mechanism by which the oxidizing environment of transformed cells promotes ATM activity and blocks cell proliferation

Genetic separation of Sae2 nuclease activity from Mre11 nuclease functions in budding yeast

Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities

Genetic separation of Sae2 nuclease activity from Mre11 nuclease functions in budding yeast

Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities

Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress

The denitrosylase S-nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S-nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhibitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox-mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox-insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T-cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein–interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku−/− mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein–interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku−/− mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847

TRIP13 Participates in Immediate-Early Sensing of DNA Strand Breaks and ATM Signaling Amplification through MRE11

Thyroid hormone receptor-interacting protein 13 (TRIP13) participates in various regulatory steps related to the cell cycle, such as the mitotic spindle assembly checkpoint and meiotic recombination, possibly by interacting with members of the HORMA domain protein family. Recently, it was reported that TRIP13 could regulate the choice of the DNA repair pathway, i.e., homologous recombination (HR) or nonhomologous end-joining (NHEJ). However, TRIP13 is recruited to DNA damage sites within a few seconds after damage and may therefore have another function in DNA repair other than regulation of the pathway choice. Furthermore, the depletion of TRIP13 inhibited both HR and NHEJ, suggesting that TRIP13 plays other roles besides regulation of choice between HR and NHEJ. To explore the unidentified functions of TRIP13 in the DNA damage response, we investigated its genome-wide interaction partners in the context of DNA damage using quantitative proteomics with proximity labeling. We identified MRE11 as a novel interacting partner of TRIP13. TRIP13 controlled the recruitment of MDC1 to DNA damage sites by regulating the interaction between MDC1 and the MRN complex. Consistently, TRIP13 was involved in ATM signaling amplification. Our study provides new insight into the function of TRIP13 in immediate-early DNA damage sensing and ATM signaling activation

Loss of ATM kinase activity leads to embryonic lethality in mice

Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis

Ancient and Recent Adaptive Evolution of Primate Non-Homologous End Joining Genes

In human cells, DNA double-strand breaks are repaired primarily by the non-homologous end joining (NHEJ) pathway. Given their critical nature, we expected NHEJ proteins to be evolutionarily conserved, with relatively little sequence change over time. Here, we report that while critical domains of these proteins are conserved as expected, the sequence of NHEJ proteins has also been shaped by recurrent positive selection, leading to rapid sequence evolution in other protein domains. In order to characterize the molecular evolution of the human NHEJ pathway, we generated large simian primate sequence datasets for NHEJ genes. Codon-based models of gene evolution yielded statistical support for the recurrent positive selection of five NHEJ genes during primate evolution: XRCC4, NBS1, Artemis, POLλ, and CtIP. Analysis of human polymorphism data using the composite of multiple signals (CMS) test revealed that XRCC4 has also been subjected to positive selection in modern humans. Crystal structures are available for XRCC4, Nbs1, and Polλ; and residues under positive selection fall exclusively on the surfaces of these proteins. Despite the positive selection of such residues, biochemical experiments with variants of one positively selected site in Nbs1 confirm that functions necessary for DNA repair and checkpoint signaling have been conserved. However, many viruses interact with the proteins of the NHEJ pathway as part of their infectious lifecycle. We propose that an ongoing evolutionary arms race between viruses and NHEJ genes may be driving the surprisingly rapid evolution of these critical genes