CtIP/Ctp1/Sae2, molecular form fit for function

Vertebrate CtIP, and its fission yeast (Ctp1), budding yeast (Sae2) and plant (Com1) orthologs have emerged as key regulatory molecules in cellular responses to DNA double strand breaks (DSBs). By modulating the nucleolytic 5′-3′ resection activity of the Mre11/Rad50/Nbs1 (MRN) DSB repair processing and signaling complex, CtIP/Ctp1/Sae2/Com1 is integral to the channeling of DNA double strand breaks through DSB repair by homologous recombination (HR). Nearly two decades since its discovery, emerging new data are defining the molecular underpinnings for CtIP DSB repair regulatory activities. CtIP homologs are largely intrinsically unstructured proteins comprised of expanded regions of low complexity sequence, rather than defined folded domains typical of DNA damage metabolizing enzymes and nucleases. A compact structurally conserved N-terminus forms a functionally critical tetrameric helical dimer of dimers (THDD) region that bridges CtIP oligomers, and is flexibly appended to a conserved C-terminal Sae2-homology DNA binding and DSB repair pathway choice regulatory hub which influences nucleolytic activities of the MRN core nuclease complex. The emerging evidence from structural, biophysical, and biological studies converges on CtIP having functional roles in DSB repair that include: 1) dynamic DNA strand coordination through direct DNA binding and DNA bridging activities, 2) MRN nuclease complex cofactor functions that direct MRN endonucleolytic cleavage of protein-blocked DSB ends and 3) acting as a protein binding hub targeted by the cell cycle regulatory apparatus, which influences CtIP expression and activity via layers of post-translational modifications, protein–protein interactions and DNA binding.     

Defective Mre11-dependent activation of Chk2 by ataxia telangiectasia mutated in colorectal carcinoma cells in response to replication-dependent DNA double strand breaks

The Mre11.Rad50.Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. We report MRN deficiency in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibited Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G2 arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers.     

The role of NBS1 in DNA double strand break repair, telomere stability, and cell cycle checkpoint control

The genomes of eukaryotic cells are under continuous assault by environmental agents and endogenous metabolic byproducts. Damage induced in DNA usually leads to a cascade of cellular events, the DNA damage response. Failure of the DNA damage response can lead to development of malignancy by reducing the efficiency and fidelity of DNA repair. The NBS1 protein is a component of the MRE11/RAD50/NBS 1 complex （MRN） that plays a critical role in the cellular response to DNA damage and the maintenance of chromosomal integrity. Mutations in the NBS1 gene are responsible for Nijmegen breakage syndrome （NBS）, a hereditary disorder that imparts an increased predisposition to development of malignancy. The phenotypic characteristics of cells isolated from NBS patients point to a deficiency in the repair of DNA double strand breaks. Here, we review the current knowledge of the role of NBS1 in the DNA damage response. Emphasis is placed on the role of NBS1 in the DNA double strand repair, modulation of the DNA damage sensing and signaling, cell cycle checkpoint control and maintenance oftelomere stability.     

Cell Cycle- and Proteasome-Dependent Formation of Etoposide-Induced Replication Protein A (RPA) or Mre11/Rad50/Nbs1 (MRN) Complex Repair Foci

In response to DNA damage, cells activate a complex protein network designed to sustain genomic integrity. Many of the proteins involved in the network form discrete repair foci, the composition of which is determined by the specific type of damage. Replication protein A (RPA) and the Mre11/Rad50/Nbs1 (MRN) complex both participate in foci and co-localize at certain types of lesions. Following etoposide (ETOP) treatment, cells form foci containing either RPA or the MRN complex, but not both. To investigate this preferential foci formation, we used cell cycle synchronization experimentation. We demonstrate that cells in S phase contain RPA foci but lack phospho-Nbs1 foci. This is consistent with RPA’s role in homologous recombination repair of DNA double-strand breaks (DSBs), the predominant form of repair during S phase. Cells synchronized at G0/G1 phase contain phospho-Nbs1 foci, consistent with the MRN complex involvement in non-homologous end joining, the predominant form of repair in G1 phase. Treatment of cells with the proteasome inhibitor MG132 dramatically reduced the percentage of cells forming phospho-Nbs1 foci but did not alter the percentage of cells containing RPA or phospho-RPA foci. ETOP induced similar amounts of damage in all phases of the cell cycle as measured by the comet assay. These data suggest that in response to DNA DSBs, cell cycle-preferred repair pathways differentially engage RPA and the MRN complex in repair foci.     

Mre11-Rad50-Nbs1 conformations and the control of sensing, signaling, and effector responses at DNA double-strand breaks

Repair and integrity of DNA ends at breaks, replication forks and telomeres are essential for life; yet, paradoxically, these responses are, in many cases, controlled by a single protein complex, Mre11-Rad50-Nbs1 (MRN). The MRN complex consists of dimers of each subunit and this heterohexamer controls key sensing, signaling, regulation, and effector responses to DNA double-strand breaks including ATM activation, homologous recombinational repair, microhomology-mediated end joining and, in some organisms, non-homologous end joining. We propose that this is possible because each MRN subunit can exist in three or more distinct states; thus, the trimer of MRN dimers can exist in a stunning 6(3) or 216 states, a number that can be expanded further when post-translational modifications are taken into account. MRN can therefore be considered as a molecular computer that effectively assesses optimal responses and pathway choice based upon its states as set by cell status and the nature of the DNA damage. This extreme multi-state concept demands a paradigm shift from striving to understand DNA damage responses in separate terms of signaling, checkpoint, and effector proteins: we must now endeavor to characterize conformational and assembly states of MRN and other DNA repair machines that couple, coordinate, and control biological outcomes. Addressing the emerging challenge of gaining a detailed molecular understanding of MRN and other multi-state dynamic DNA repair machines promises to provide opportunities to develop master keys for controlling cell biology with probable impacts on therapeutic interventions.     

Rad17 recruits the MRE11‐RAD50‐NBS1 complex to regulate the cellular response to DNA double‐strand breaks

The MRE11‐RAD50‐NBS1 (MRN) complex is essential for the detection of DNA double‐strand breaks (DSBs) and initiation of DNA damage signaling. Here, we show that Rad17, a replication checkpoint protein, is required for the early recruitment of the MRN complex to the DSB site that is independent of MDC1 and contributes to ATM activation. Mechanistically, Rad17 is phosphorylated by ATM at a novel Thr622 site resulting in a direct interaction of Rad17 with NBS1, facilitating recruitment of the MRN complex and ATM to the DSB, thereby enhancing ATM signaling. Repetition of these events creates a positive feedback for Rad17‐dependent activation of MRN/ATM signaling which appears to be a requisite for the activation of MDC1‐dependent MRN complex recruitment. A point mutation of the Thr622 residue of Rad17 leads to a significant reduction in MRN/ATM signaling and homologous recombination repair, suggesting that Thr622 phosphorylation is important for regulation of the MRN/ATM signaling by Rad17. These findings suggest that Rad17 plays a critical role in the cellular response to DNA damage via regulation of the MRN/ATM pathway.     

Histone tails: Directing the chromatin response to DNA damage

Considerable energetic investment is devoted to altering large stretches of chromatin adjacent to DNA double strand breaks (DSBs). Immediately ensuing DSB formation, a myriad of histone modifications are elicited to create a platform for inducible and modular assembly of DNA repair protein complexes in the vicinity of the DNA lesion. This complex signaling network is critical to repair DNA damage and communicate with cellular processes that occur in cis and in trans to the genomic lesion. Failure to properly execute DNA damage inducible chromatin changes is associated with developmental abnormalities, immunodeficiency, and malignancy in humans and in genetically engineered mouse models. This review will discuss current knowledge of DNA damage responsive histone changes that occur in mammalian cells, highlighting their involvement in the maintenance of genome integrity.     

Ataxia-telangiectasia-like disorder (ATLD)-its clinical presentation and molecular basis

Comparison of the clinical and cellular phenotypes of different genomic instability syndromes provides new insights into functional links in the complex network of the DNA damage response. A prominent example of this principle is provided by examination of three such disorders: ataxia-telangiectasia (A-T) caused by lack or inactivation of the ATM protein kinase, which mobilises the cellular response to double strand breaks in the DNA; ataxia-telangiectasia-like disease (ATLD), a result of deficiency of the human Mre11 protein; and the Nijmegen breakage syndrome (NBS), which represents defective Nbs1 protein. Mre11 and Nbs1 are members of the Mre11/Rad50/Nbs1 (MRN) protein complex. MRN and its individual components are involved in different responses to cellular damage induced by ionising radiation and radiomimetic chemicals, including complexing with chromatin and with other damage response proteins, formation of radiation-induced foci, and the induction of different cell cycle checkpoints. The phosphorylation of Nbs1 by ATM would indicate that ATM acts upstream of the MRN complex. Consistent with this were the suggestions that ATM could be activated in the absence of fully functional Nbs1 protein. In contrast, the regulation of some ATM target proteins, e.g. Smc1 requires the MRN complex as well as ATM. Nbs1 may, therefore, be both a substrate for ATM and a mediator of ATM function. Recent studies that indicate a requirement of the MRN complex for proper ATM activation suggest that the relationship between ATM and the MRN complex in the DNA damage response is yet to be fully determined. Despite the fact that both Mre11 and Nbs1 are part of the same MRN complex, deficiency in either protein in humans does not lead to the same clinical picture. This suggests that components of the complex may also act separately.     

Rad17, the clamp loader that loads more than clamps

Rad17 is best known as a checkpoint clamp loader in the activation of ATR kinase signaling. A new study in The EMBO Journal suggests that it also plays a role in initial recruitment of the MRN complex to sites of DNA double‐strand breaks, thereby promoting early ATM checkpoint responses and homologous recombination repair.     

NBS1在DNA断裂损伤反应和维持端粒稳定中的作用

NBS1作为MRE11/RAD50/NBS1复合物的组分之一,是细胞应答DNA损伤的一个关键蛋白质,在DNA双链断裂修复和维持基因组稳定中发挥重要的作用。端粒是染色体末端由DNA重复序列和蛋白质构成的复合体,其独特结构与DNA双链断裂非常相似。最近几年的研究发现NBS1与端粒也有着十分密切的联系。综述了NBS1在DNA损伤反应中的作用,并探讨NBS1参与维持端粒稳定中的分子机制。     

Tip60: Connecting chromatin to DNA damage signaling

Cells are constantly exposed to genotoxic events that can damage DNA. To counter this, cells have evolved a series of highly conserved DNA repair pathways to maintain genomic integrity. The ATM protein kinase is a master regulator of the DNA double-strand break (DSB) repair pathway. DSBs activate ATM’s kinase activity, promoting the phosphorylation of proteins involved in both checkpoint activation and DNA repair. Recent work has revealed that two DNA damage response proteins, the Tip60 acetyltransferase and the mre11-rad50-nbs1 (MRN) complex, co-operate in the activation of ATM in response to DSBs. MRN functions to target ATM and the Tip60 acetyltransferase to DSBs. Tip60’s chromodomain then interacts with histone H3 trimethylated on lysine 9, activating Tip60’s acetyltransferase activity and stimulating the subsequent acetylation and activation of ATM’s kinase activity. These results underscore the importance of chromatin structure in regulating DNA damage signaling and emphasize how histone modifications co-ordinate DNA repair. In addition, human tumors frequently exhibit altered patterns of histone methylation. This rewriting of the histone methylation code in tumor cells may impact the efficiency of DSB repair, increasing genomic instability and contributing to the initiation and progression of cancer.     

Solving the RIDDLE of 53BP1 recruitment to sites of damage

The cellular response to DNA double strand breaks is a complex, integrated network of pathways, coordinated by the PI-3-kinase-like family of kinases, which includes ATM, ATR and DNA-PK, that function to preserve the integrity of the genome. Mutations in genes that control these pathways are associated with increased genomic instability, neurodegeneration, immunodeficiency, premature aging and tumour predisposition. Indeed a significant proportion of our understanding regarding the mechanisms controlling DNA double strand break (DSB) repair has come from the study of cells derived from patients with inherited mutations in these genes. The discovery of the E3 ubiquitin ligase, RNF8, as a regulator of DNA DSB repair has brought to light a critical role for the ubiquitin system in regulating the cellular DSBs. Recently, identification of mutations in a second E3 ubiquitin ligase, RNF168, as the underlying genetic cause of the DNA repair deficiency disorder, RIDDLE syndrome, has provided the first link between ubiquitin-dependent DSB repair and immune system development in man. The finding that RNF168 functions downstream of RNF8 to orchestrate the recruitment of repair proteins, such as BRCA1 and 53BP1, to sites of DNA damage suggests that these two E3 ligases define a ubiquitylation cascade that regulates the spatial relocalisation of DSB repair proteins.     

Distinct Roles of the ATR Kinase and the Mre11-Rad50-Nbs1 Complex in the Maintenance of Chromosomal Stability in Arabidopsis

Signaling of chromosomal DNA breaks is of primary importance for initiation of repair and, thus, for global genomic stability. Although the Mre11-Rad50-Nbs1 (MRN) complex is the first sensor of double-strand breaks, its role in double-strand break (DSB) signaling is not fully understood. We report the absence of γ-ray–induced, ATM/ATR-dependent histone H2AX phosphorylation in Arabidopsis thaliana rad50 and mre11 mutants, confirming that the MRN complex is required for H2AX phosphorylation by the ATM and ATR kinases in response to irradiation-induced DSB in Arabidopsis. rad50 and mre11 mutants spontaneously activate a DNA damage response, as shown by the presence of γ-H2AX foci and activation of cell cycle arrest in nonirradiated plants. This response is ATR dependent as shown both by the absence of these spontaneous foci and by the wild-type mitotic indices of double rad50 atr and mre11 atr plants. EdU S-phase labeling and fluorescence in situ hybridization analysis using specific subtelomeric probes point to a replicative S-phase origin of this chromosome damage in the double mutants and not to telomere destabilization. Thus, the data presented here show the exclusive involvement of ATR in DNA damage signaling in MRN mutants and provide evidence for a role for ATR in the avoidance of S-phase DNA damage.     

XPD-dependent activation of apoptosis in response to triplex-induced DNA damage

DNA sequences capable of forming triplexes are prevalent in the human genome and have been found to be intrinsically mutagenic. Consequently, a balance between DNA repair and apoptosis is critical to counteract their effect on genomic integrity. Using triplex-forming oligonucleotides to synthetically create altered helical distortions, we have determined that pro-apoptotic pathways are activated by the formation of triplex structures. Moreover, the TFIIH factor, XPD, occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. Here, we show that triplexes are capable of inducing XPD-independent double strand breaks, which result in the formation of γH2AX foci. XPD was subsequently recruited to the triplex-induced double strand breaks and co-localized with γH2AX at the damage site. Furthermore, phosphorylation of H2AX tyrosine 142 was found to stimulate the signaling pathway of XPD-dependent apoptosis. We suggest that this mechanism may play an active role in minimizing genomic instability induced by naturally occurring noncanonical structures, perhaps protecting against cancer initiation.     

Telomeres and the DNA damage response: why the fox is guarding the henhouse

DNA double strand breaks (DSBs) are repaired by an extensive network of proteins that recognize damaged DNA and catalyze its repair. By virtue of their similarity, the normal ends of linear chromosomes and internal DNA DSBs are both potential substrates for DSB repair enzymes. Thus, telomeres, specialized nucleo-protein complexes that cap chromosomal ends, serve a critical function to differentiate themselves from internal DNA strand breaks, and as a result prevent genomic instability that can result from their inappropriate involvement in repair reactions. Telomeres that become critically short due to failure of telomere maintenance mechanisms, or which become dysfunctional by loss of telomere binding proteins, elicit extensive checkpoint responses that in normal cells blocks proliferation. In this situation, the DNA DSB repair machinery plays a major role in responding to these "damaged" telomeres - creating chromosome fusions or capturing telomeres from other chromosomes in an effort to rid the cell of the perceived damage. However, a surprising aspect of telomere maintenance is that many of the same proteins that facilitate this repair of damaged telomeres are also necessary for their proper integrity. Here, we review recent work defining the roles for DSB repair machinery in telomere maintenance and in response to telomere dysfunction.     

Contribution of DNA repair and cell cycle checkpoint arrest to the maintenance of genomic stability

DNA damage response mechanisms encompass pathways of DNA repair, cell cycle checkpoint arrest and apoptosis. Together, these mechanisms function to maintain genomic stability in the face of exogenous and endogenous DNA damage. ATM is activated in response to double strand breaks and initiates cell cycle checkpoint arrest. Recent studies in human fibroblasts have shown that ATM also regulates a mechanism of end-processing that is required for a component of double strand break repair. Human fibroblasts rarely undergo apoptosis after ionising radiation and, therefore, apoptosis is not considered in our review. The dual function of ATM raises the question as to how the two processes, DNA repair and checkpoint arrest, interplay to maintain genomic stability. In this review, we consider the impact of ATM's repair and checkpoint functions to the maintenance of genomic stability following irradiation in G2. We discuss evidence that ATM's repair function plays little role in the maintenance of genomic stability following exposure to ionising radiation. ATM's checkpoint function has a bigger impact on genomic stability but strikingly the two damage response pathways co-operate in a more than additive manner. In contrast, ATM's repair function is important for survival post irradiation.     

DNA-dependent Protein Kinase Regulates DNA End Resection in Concert with Mre11-Rad50-Nbs1 (MRN) and Ataxia Telangiectasia-mutated (ATM)

The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice.     

Requirement of the MRN complex for ATM activation by DNA damage

The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD.     

Human induced pluripotent cells resemble embryonic stem cells demonstrating enhanced levels of DNA repair and efficacy of nonhomologous end-joining

To maintain the integrity of the organism, embryonic stem cells (ESC) need to maintain their genomic integrity in response to DNA damage. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and can have disastrous consequences if not repaired correctly, leading to cell death, genomic instability and cancer. How human ESC (hESC) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. Adult somatic cells can be induced to "dedifferentiate" into induced pluripotent stem cells (iPSC) and reprogram into cells of all three germ layers. Whether iPSC have reprogrammed the DNA damage response is a critical question in regenerative medicine. Here, we show that hESC demonstrate high levels of endogenous reactive oxygen species (ROS) which can contribute to DNA damage and may arise from high levels of metabolic activity. To potentially counter genomic instability caused by DNA damage, we find that hESC employ two strategies: First, these cells have enhanced levels of DNA repair proteins, including those involved in repair of DSBs, and they demonstrate elevated nonhomologous end-joining (NHEJ) activity and repair efficacy, one of the main pathways for repairing DSBs. Second, they are hypersensitive to DNA damaging agents, as evidenced by a high level of apoptosis upon irradiation. Importantly, iPSC, unlike the parent cells they are derived from, mimic hESC in their ROS levels, cell cycle profiles, repair protein expression and NHEJ repair efficacy, indicating reprogramming of the DNA repair pathways. Human iPSC however show a partial apoptotic response to irradiation, compared to hESC. We suggest that DNA damage responses may constitute important markers for the efficacy of iPSC reprogramming.     

Focus: 50 Years of DNA Repair: The Yale Symposium Reports: Triplex-Induced DNA Damage Response

Cellular DNA damage response is critical to preserving genomic integrity following exposure to genotoxic stress. A complex series of networks and signaling pathways become activated after DNA damage and trigger the appropriate cellular response, including cell cycle arrest, DNA repair, and apoptosis. The response elicited is dependent upon the type and extent of damage sustained, with the ultimate goal of preventing propagation of the damaged DNA. A major focus of our studies is to determine the cellular pathways involved in processing damage induced by altered helical structures, specifically triplexes. Our lab has demonstrated that the TFIIH factor XPD occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. We have shown that XPD co-localizes with γH2AX, and its presence is required for the phosphorylation of H2AX tyrosine142, which stimulates the signaling pathway to recruit pro-apoptotic factors to the damage site. Herein, we examine the cellular pathways activated in response to triplex formation and discuss our finding that suggests that XPD-dependent apoptosis plays a role in preserving genomic integrity in the presence of excessive structurally induced DNA damage.