In vertebrate cells, the DNA damage response is controlled by three related kinases: ATM, ATR, and DNA-PK. It has been 20 years since the cloning of ATR, the last of the three to be identified. During this time, our understanding of how these kinases regulate DNA repair and associated events has grown profoundly, although major questions remain unanswered. Here, we provide a historical perspective of their discovery and discuss their established functions in sensing and responding to genotoxic stress. We also highlight what is known regarding their structural similarities and common mechanisms of regulation, as well as emerging non-canonical roles and how our knowledge of ATM, ATR, and DNA-PK is being translated to benefit human health.

For many years, much research has focused on understanding how cells maintain genome integrity despite DNA being constantly challenged by factors of both endogenous and exogenous nature. DNA double-strand breaks (DSBs) are the most deleterious DNA lesions, and if left unrepaired or repaired incorrectly, a single DSB can trigger genome instability or even cell death1. Therefore, any DSB has to be recognized and repaired by processes encompassed within the DNA damage response (DDR). Notably, while the ends of mammalian linear chromosomes naturally resemble DSBs, their structure and association with the so-called “Shelterin” complex normally makes them invisible to the DDR2.As soon as a DSB is formed, it is sensed and directly bound by the Ku70-Ku80 and/or MRE11-NBS1-RAD50 protein complexes, which recruit and activate the DDR kinases DNA-PKcs and ATM, respectively. The first steps in the DDR to DSBs are followed by cascades of events involving protein post-translational modifications (PTMs) and formation of large protein assemblies at DSB sites known as ionizing radiation-induced foci (IRIF)3. Protein phosphorylation and ubiquitylation are at the heart of these signaling processes3. For example, following recruitment of the DDR mediator protein MDC1 to the phospho-epitope created by ATM and DNA-PKcs on variant histone H2AX, MDC1 is itself phosphorylated by ATM on multiple serines and threonines4. MDC1 phosphorylation on a group of threonines near its N-terminus and conforming to the consensus TQXF generates binding sites for the FHA domain of E3-ubiquitin ligase RNF85,6. Together with the E2-conjugating enzyme UBC13, RNF8, and another E3 ligase, RNF168, trigger formation of mainly lysine 63-linked ubiquitin adducts in DSB-proximal chromatin, promoting recruitment of downstream factors necessary for DNA repair, such as the RAP80-Abraxas-BRCA1 complex and 53BP13.Significantly, the full DDR happens only in interphase cells, whereas if mitotic cells sustain DSBs, the process appears to be blocked at the stage of RNF8 recruitment, resulting in IRIF devoid of detectable ubiquitin conjugates7. Consequently, 53BP1 and BRCA1 are not recruited to IRIF during mitosis. Even more strikingly, although RNF8 and RNF168 are associated with mitotic IRIF in anaphase, hyperphosphorylated 53BP1 remains excluded from chromatin until cells progress into G1 phase7. Based on these findings, it was hypothesized that mitosis-specific PTMs on RNF8 and 53BP1 might preclude formation of repair-competent IRIF7. However, the precise mechanistic explanation of the “interrupted” DDR in mitosis remained to be unravelled.A recent study published in Science by the group of Daniel Durocher addressed the question of how full IRIF assembly and DSB repair are prevented in mitotic cells8. First, Orthwein et al. focused on the mechanism that abrogates RNF8 recruitment to DSBs during mitosis. They demonstrated that CDK1-dependent mitosis-specific phosphorylation of RNF8 on T198 abolished interaction between RNF8 and its target phospho-TQXF motifs in MDC1. This important finding was somewhat surprising, given that MDC1 binding is mediated by the RNF8 FHA domain5,6 and T198 is located some distance away from this domain. It will thus be interesting to see how T198 phosphorylation abrogates MDC1 binding, for example via T198 being juxtaposed to the FHA domain in the RNF8 3D structure, through phosphorylated T198 docking with the phospho-binding region of the FHA domain, or via another mechanism. In this regard, we note that T198 is part of an STP motif, which upon modification by CDK1 could constitute a priming site for PLK1 kinase9. Thus, T198 phosphorylation might be followed by PLK1-mediated RNF8 phosphorylation. Interestingly, certain sites in RNF8 conform to the PLK1 consensus motif, with those at T39 and T316 being evolutionarily conserved in vertebrates. Moreover, T39 is located in the FHA domain, close to R42, mutation of which abolishes RNF8 interaction with MDC15,6. It would therefore be worthwhile mutating these potential PLK1 sites and establishing whether this affects mitotic control of RNF8 binding to MDC1.After identifying T198 as critical for preventing RNF8 recruitment to DSBs during mitosis, Orthwein et al. observed that, while mutating this residue to alanine restored recruitment of RNF8 (and BRCA1) to mitotic IRIF, 53BP1 still remained excluded from DSB sites. This prompted the authors to look for mitosis-specific PTMs of 53BP1 by mass spectrometry, leading to the discovery of two novel phosphosites mapped to the recently described ubiquitin-dependent recruitment (UDR) motif, which mediates binding to ubiquitylated H2A and is required for 53BP1 IRIF formation10. Notably, the same residues, T1609 and S1618, were also identified by Chowdhury and colleagues11 as target sites for the PP4C/R3β phosphatase. This group showed that T1609 and S1618 must be dephosphorylated for 53BP1 to form IRIF. In accord with these findings, Orthwein et al. established that when T1609 and S1618 were mutated to alanines, the ensuing “53BP1-TASA” protein was recruited to sites of DNA damage during mitosis in cells expressing RNF8-T198A. Moreover, unlike normal cells, cells co-expressing RNF8-T198A and 53BP1-TASA carried out DSB joining reactions during mitosis and were extremely hypersensitive to ionizing radiation (IR). The authors also found that, following irradiation in mitosis, cells carrying these mutant RNF8 and 53BP1 proteins displayed increased rates of kinetochore-positive micronucleus formation, suggesting mis-segregation of full chromosomes. In addition, chromosomes in these cells were prone to sister telomere fusions, thereby helping to explain their elevated levels of aneuploidy and IR hypersensitivity.The research described above has not only revealed how DSB repair is suppressed in mitosis but has also established that this suppression is biologically important. Orthwein et al. propose that, as mitotic telomeres become “underprotected” when mitosis is prolonged upon stress12, this could lead to telomere fusion if DNA end-joining pathway is active. The suppression of DSB signaling and repair mediated by RNF8 and 53BP1 mitotic phosphorylation therefore probably evolved as a mechanism to mitigate this threat to genome stability. A key question that still remains is why mitotic telomeres become underprotected in the first place? Also, what features in telomere structure or replication and segregation processes make it more beneficial for the cells to keep chromosome ends less protected at the cost of inhibiting the DDR during mitosis? Finally, given that cancers often harbor cell cycle and/or DDR defects1, it will be of interest to see whether defective mitotic control of DSB repair might play a role in tumor evolution, or could provide opportunities for developing better anti-cancer therapies.

Chromothripsis is an emerging phenomenon that results in chromosome rearrangements in tumour cells. This Review discusses the possible mechanisms underlying this process and its implications for cancer biology and in the clinic.

Following the formation of a DNA double-strand break (DSB), cells activate the DNA-damage response and recruit a number of proteins to the lesion. Some of these proteins are modified by the attachment of small ubiquitin-related modifier (SUMO). Here, SUMO1, SUMO2 and SUMO3 are shown to accumulate at DSB sites in mammalian cells. SUMO1 and SUMO2/3 accrual requires the E3 ligase enzymes PIAS4 and PIAS1, which promote DSB repair.

DNA double-strand breaks (DSBs) are repaired by two principal mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR)1. HR is the most accurate DSB repair mechanism but is generally restricted to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as a repair template2, 3, 4, 5. By contrast, NHEJ operates throughout the cell cycle but assumes most importance in G1 (refs 4, 6). The choice between repair pathways is governed by cyclin-dependent protein kinases (CDKs)2, 3, 5, 7, with a major site of control being at the level of DSB resection, an event that is necessary for HR but not NHEJ, and which takes place most effectively in S and G2 (refs 2, 5). Here we establish that cell-cycle control of DSB resection in Saccharomyces cerevisiae results from the phosphorylation by CDK of an evolutionarily conserved motif in the Sae2 protein. We show that mutating Ser 267 of Sae2 to a non-phosphorylatable residue causes phenotypes comparable to those of a sae2Δ null mutant, including hypersensitivity to camptothecin, defective sporulation, reduced hairpin-induced recombination, severely impaired DNA-end processing and faulty assembly and disassembly of HR factors. Furthermore, a Sae2 mutation that mimics constitutive Ser 267 phosphorylation complements these phenotypes and overcomes the necessity of CDK activity for DSB resection. The Sae2 mutations also cause cell-cycle-stage specific hypersensitivity to DNA damage and affect the balance between HR and NHEJ. These findings therefore provide a mechanistic basis for cell-cycle control of DSB repair and highlight the importance of regulating DSB resection.

In the S and G2 phases of the cell cycle, DNA double-strand breaks (DSBs) are processed into single-stranded DNA, triggering ATR-dependent checkpoint signalling and DSB repair by homologous recombination. Previous work has implicated the MRE11 complex in such DSB-processing events. Here, we show that the human CtIP (RBBP8) protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in the S and G2 cell-cycle phases. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of replication protein A (RPA) and the protein kinase ATR to DSBs, and for the ensuing ATR activation. Furthermore, we establish that CtIP physically and functionally interacts with the MRE11 complex, and that both CtIP and MRE11 are required for efficient homologous recombination. Finally, we reveal that CtIP has sequence homology with Sae2, which is involved in MRE11-dependent DSB processing in yeast. These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signalling and homologous recombination.

Abstract

Posttranslational modifications of the histone octamer play important roles in regulating responses to DNA damage. Here, we reveal that Saccharomyces cerevisiae Rtt109p promotes genome stability and resistance to DNA-damaging agents, and that it does this by functionally cooperating with the histone chaperone Asf1p to maintain normal chromatin structure. Furthermore, we show that, as for Asf1p, Rtt109p is required for histone H3 acetylation on lysine 56 (K56) in vivo. Moreover, we show that Rtt109p directly catalyzes this modification in vitro in a manner that is stimulated by Asf1p. These data establish Rtt109p as a member of a new class of histone acetyltransferases and show that its actions are critical for cell survival in the presence of DNA damage during S phase.

Ubiquitylation and sumoylation, the covalent attachment of the polypeptides ubiquitin and SUMO, respectively, to target proteins, are pervasive mechanisms for controlling cellular functions. Here, we summarize the key steps and enzymes involved in ubiquitin and SUMO conjugation and provide an overview of how they are crucial for maintaining genome stability. Specifically, we review research that has revealed how ubiquitylation and sumoylation regulate and coordinate various pathways of DNA damage recognition, signaling, and repair at the biochemical, cellular, and whole-organism levels. In addition to providing key insights into the control and importance of DNA repair and associated processes, such work has established paradigms for regulatory control that are likely to extend to other cellular processes and that may provide opportunities for better understanding and treatment of human disease.

•The deubiquitylating enzyme USP4 promotes homologous recombination-mediated repair•USP4 regulates recruitment and/or association dynamics of CtIP at DNA damage sites•Auto-deubiquitylation of USP4 stimulates HR events and interactions with CtIP/MRN•Ubiquitin adducts might regulate USP-enzyme interactions/function more commonlyRepair of DNA double-strand breaks is crucial for maintaining genome integrity and is governed by post-translational modifications such as protein ubiquitylation. Here, we establish that the deubiquitylating enzyme USP4 promotes DNA-end resection and DNA repair by homologous recombination. We also report that USP4 interacts with CtIP and the MRE11-RAD50-NBS1 (MRN) complex and is required for CtIP recruitment to DNA damage sites. Furthermore, we show that USP4 is ubiquitylated on multiple sites including those on cysteine residues and that deubiquitylation of these sites requires USP4 catalytic activity and is required for USP4 to interact with CtIP/MRN and to promote CtIP recruitment and DNA repair. Lastly, we establish that regulation of interactor binding by ubiquitylation occurs more generally among USP-family enzymes. Our findings thus identify USP4 as a novel DNA repair regulator and invoke a model in which ubiquitin adducts regulate USP enzyme interactions and functions.Download high-res image (200KB)Download full-size image

The regulatory networks of the DNA damage response (DDR) encompass many proteins and posttranslational modifications. Here, we use mass spectrometry-based proteomics to analyze the systems-wide response to DNA damage by parallel quantification of the DDR-regulated phosphoproteome, acetylome, and proteome. We show that phosphorylation-dependent signaling networks are regulated more strongly compared to acetylation. Among the phosphorylated proteins identified are many putative substrates of DNA-PK, ATM, and ATR kinases, but a majority of phosphorylated proteins do not share the ATM/ATR/DNA-PK target consensus motif, suggesting an important role of downstream kinases in amplifying DDR signals. We show that the splicing-regulator phosphatase PPM1G is recruited to sites of DNA damage, while the splicing-associated protein THRAP3 is excluded from these regions. Moreover, THRAP3 depletion causes cellular hypersensitivity to DNA-damaging agents. Collectively, these data broaden our knowledge of DNA damage signaling networks and highlight an important link between RNA metabolism and DNA repair.Highlights► Global quantification of DDR-regulated phosphoproteome, acetylome, and proteome ► Phosphorylation is regulated more robustly than acetylation after DNA damage ► Much DDR-induced phosphorylation depends on non-ATM/ATR/DNA-PK kinases ► THRAP3 is phosphorylated and excluded from sites of DNA damage

DNA double-strand break (DSB) signaling and repair are critical for cell viability, and rely on highly coordinated pathways whose molecular organization is still incompletely understood. Here, we show that heterogeneous nuclear ribonucleoprotein U-like (hnRNPUL) proteins 1 and 2 play key roles in cellular responses to DSBs. We identify human hnRNPUL1 and -2 as binding partners for the DSB sensor complex MRE11-RAD50-NBS1 (MRN) and demonstrate that hnRNPUL1 and -2 are recruited to DNA damage in an interdependent manner that requires MRN. Moreover, we show that hnRNPUL1 and -2 stimulate DNA-end resection and promote ATR-dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents. Finally, we establish that hnRNPUL1 and -2 function downstream of MRN and CtBP-interacting protein (CtIP) to promote recruitment of the BLM helicase to DNA breaks. Collectively, these results provide insights into how mammalian cells respond to DSBs.Graphical AbstractDownload high-res image (378KB)Download full-size imageHighlights► hnRNPUL proteins associate with the DSB sensor complex MRN ► hnRNPUL proteins display both exclusion from and MRN-dependent recruitment to DSBs ► hnRNPUL proteins stimulate DSB resection, signaling, and repair ► hnRNPUL proteins promote BLM recruitment to sites of DNA damage

•Contrary to the findings of Wang et al. (2013), TopBP1 does not maintain BLM levels•BLM phosphorylation on Ser304, not Ser338, is required for TopBP1 binding•Disrupting BLM-TopBP1 binding causes chromosome instability•Crucial residues mediating TopBP1-MDC1 and BLM-TOP3A/RMI interactions identifiedThe Bloom syndrome helicase BLM and topoisomerase-IIβ-binding protein 1 (TopBP1) are key regulators of genome stability. It was recently proposed that BLM phosphorylation on Ser338 mediates its interaction with TopBP1, to protect BLM from ubiquitylation and degradation (Wang et al., 2013). Here, we show that the BLM-TopBP1 interaction does not involve Ser338 but instead requires BLM phosphorylation on Ser304. Furthermore, we establish that disrupting this interaction does not markedly affect BLM stability. However, BLM-TopBP1 binding is important for maintaining genome integrity, because in its absence cells display increased sister chromatid exchanges, replication origin firing and chromosomal aberrations. Therefore, the BLM-TopBP1 interaction maintains genome stability not by controlling BLM protein levels, but via another as-yet undetermined mechanism. Finally, we identify critical residues that mediate interactions between TopBP1 and MDC1, and between BLM and TOP3A/RMI1/RMI2. Taken together, our findings provide molecular insights into a key tumor suppressor and genome stability network.Download high-res image (100KB)Download full-size image