DNA Double-strand Breaks Signaling Research Articles

ATP puts the brake on DNA double-strand break repair: a new study shows that ATP switches the Mre11-Rad50-Nbs1 repair factor between signaling and processing of DNA ends.

DNA double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage and can result in cell inviability or chromosomal aberrations. The Mre11-Rad50-Nbs1 (MRN) ATPase-nuclease complex is a central player in the cellular response to DSBs and is implicated in the sensing and nucleolytic processing of DSBs, as well as in DSB signaling by activating the cell cycle checkpoint kinase ATM. ATP binding to Rad50 switches MRN from an open state with exposed Mre11 nuclease sites to a closed state with partially buried nuclease sites. The functional meaning of this switch remained unclear. A new study shows that ATP binding to Rad50 promotes DSB recognition, tethering, and ATM activation, while ATP hydrolysis opens the nuclease active sites to promote processing of DSBs. MRN thus emerges as functional switch that may coordinate the temporal transition from signaling to processing of DSBs.

The Human SRCAP Chromatin Remodeling Complex Promotes DNA-End Resection

Repair of DNA double-strand breaks (DSBs) byhomologous recombination requires 5'-3' resection of the DSB ends. In vertebrates, DSB resection is initiated by the collaborative action of CtIP and the MRE11-RAD50-NBS1 (MRN) complex. However, how this process occurs within the context of chromatin is still not well understood. Here we identify the human SRCAP chromatin remodeling complex as a factor that promotes CtIP-dependent DNA-end resection. We show that SRCAP, which is mutated in Floating-Harbor syndrome, confers resistance to DNA damage-inducing agents and is recruited to DSBs. Moreover, we demonstrate that SRCAP is required for DNA-end resection, and thereby for recruitment of RPA and RAD51 to DSBs, and for the ensuing homologous recombination. Finally, we reveal that SRCAP forms a complex with CtIP and promotes accumulation of CtIP at DSBs through a mechanism involving its ATPase activity. Our study implicates the human SRCAP chromatin remodeling complex as a novel regulator of DNA damage responses that orchestrates proper signaling and repair of DSBs in the context of chromatin.

SETD2 is required for DNA double-strand break repair and activation of the p53-mediated checkpoint.

Histone modifications establish the chromatin states that coordinate the DNA damage response. In this study, we show that SETD2, the enzyme that trimethylates histone H3 lysine 36 (H3K36me3), is required for ATM activation upon DNA double-strand breaks (DSBs). Moreover, we find that SETD2 is necessary for homologous recombination repair of DSBs by promoting the formation of RAD51 presynaptic filaments. In agreement, SETD2-mutant clear cell renal cell carcinoma (ccRCC) cells displayed impaired DNA damage signaling. However, despite the persistence of DNA lesions, SETD2-deficient cells failed to activate p53, a master guardian of the genome rarely mutated in ccRCC and showed decreased cell survival after DNA damage. We propose that this novel SETD2-dependent role provides a chromatin bookmarking instrument that facilitates signaling and repair of DSBs. In ccRCC, loss of SETD2 may afford an alternative mechanism for the inactivation of the p53-mediated checkpoint without the need for additional genetic mutations in TP53.DOI: http://dx.doi.org/10.7554/eLife.02482.001.

BCL10 regulates RNF8/RNF168-mediated ubiquitination in the DNA damage response

Timely and proper cellular response to DNA damage is essential for maintenance of genome stability and integrity. B-cell lymphoma/leukemia 10 (BCL10) facilitates ubiquitination of NEMO in the cytosol, activating NFκB signaling. Translocation and/or point mutations of BCL10 associate with mucosa-associated lymphoid tissue lymphomas and other malignancies. However, the mechanisms by which the resulting aberrant expression of BCL10 leads to cellular oncogenesis are poorly understood. In this report, we found that BCL10 in the nucleus is enriched at the DNA damage sites in an ATM- and RNF8-dependent manner. ATM-dependent phosphorylation of BCL10 promotes its interaction with and presentation of UBC13 to RNF8, and RNF8-mediated ubiquitination of BCL10 enhances binding of BCL10 and UBC13 to RNF168. This allows mono-ubiquitination on H2AX by RNF168 and further poly-ubiquitination by the RNF8/RNF168-containing complex. Depletion of BCL10 compromised homology recombination-mediated DNA double-strand break (DSB) repair because of insufficient recruitment of BRCA1, RAD51, and the ubiquitinated DNA damage response factors. Taken together, our results demonstrate a novel function of BCL10 in delivering UBC13 to RNF8/RNF168 to regulate ubiquitination-mediated DSB signaling and repair.

ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling

The Mre11-Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP-driven states regulate the sensing, processing and signaling of DNA double-strand breaks are largely unknown. Here we design structure-based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP-driven movements within the catalytic domains. With this strategy we identify Rad50 separation-of-function mutants that either promote or destabilize the ATP-bound state. Crystal structures, X-ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP-induced 'closed' conformation promotes DNA end binding and end tethering, while hydrolysis-induced opening is essential for DNA resection. Reducing the stability of the ATP-bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double-strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11-Rad50-Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure-based mutational analyses. These collective results suggest that ATP-dependent Rad50 conformations switch the Mre11-Rad50 complex between DNA tethering, ATM signaling, and 5' strand resection, revealing molecular mechanisms regulating responses to DNA double-strand breaks.

Double-strand break repair: 53BP1 comes into focus

DNA double-strand break (DSB) signalling and repair is crucial to preserve genomic integrity and maintain cellular homeostasis. p53-binding protein 1 (53BP1) is an important regulator of the cellular response to DSBs that promotes the end-joining of distal DNA ends, which is induced during V(D)J and class switch recombination as well as during the fusion of deprotected telomeres. New insights have been gained into the mechanisms underlying the recruitment of 53BP1 to damaged chromatin and how 53BP1 promotes non-homologous end-joining-mediated DSB repair while preventing homologous recombination. From these studies, a model is emerging in which 53BP1 recruitment requires the direct recognition of a DSB-specific histone code and its influence on pathway choice is mediated by mutual antagonism with breast cancer 1 (BRCA1).

DNA Double-Strand Break Repair Pathway Choice Is Directed by Distinct MRE11 Nuclease Activities

MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by nonhomologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors, we examined repair pathway choice at DSBs generated in G2 following radiation exposure. While nuclease inhibition impairs radiation-induced replication protein A (RPA) chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, while exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exonuclease and EXO1/BLM bidirectional resection toward and away from the DNA end, which commits to HR.

Mutations of the Huntington’s Disease Protein Impact on the ATM-Dependent Signaling and Repair Pathways of the Radiation-Induced DNA Double-Strand Breaks: Corrective Effect of Statins and Bisphosphonates

Huntington's disease (HD) is a neurodegenerative syndrome caused by mutations of the IT15 gene encoding for the huntingtin protein. Some research groups have previously shown that HD is associated with cellular radiosensitivity in quiescent cells. However, there is still no mechanistic model explaining such specific clinical feature. Here, we examined the ATM-dependent signaling and repair pathways of the DNA double-strand breaks (DSB), the key damage induced by ionizing radiation, in human HD skin fibroblasts. Early after irradiation, quiescent HD fibroblasts showed an abnormally low rate of recognized DSB managed by non-homologous end-joining reflected by a low yield of nuclear foci formed by phosphorylated H2AX histones and by 53BP1 protein. Furthermore, HD cells elicited a significant but moderate yield of unrepaired DSB 24h after irradiation. Irradiated HD cells also presented a delayed nucleo-shuttling of phosphorylated forms of the ATM kinase, potentially due to a specific binding of ATM to mutated huntingtin in the cytoplasm. Our results suggest that HD belongs to the group of syndromes associated with a low but significant defect of DSB signaling and repair defect associated with radiosensitivity. A combination of biphosphonates and statins complements these impairments by facilitating the nucleo-shuttling of ATM, increasing the yield of recognized and repaired DSB.

The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells.

Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.

53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark

53BP1 (TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains enigmatic since its relocalization involves recognition of H4 Lys20 (H4K20) methylation by its Tudor domain. Here we elucidate how 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mono-nucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitylated on Lys15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a C-terminal extension, termed the ubiquitylation-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone “code” produced by DSB signaling.

Sodium tungstate modulates ATM function upon DNA damage

Both radiotherapy and most effective chemotherapeutic agents induce different types of DNA damage. Here we show that tungstate modulates cell response to DNA damaging agents. Cells treated with tungstate were more sensitive to etoposide, phleomycin and ionizing radiation (IR), all of which induce DNA double-strand breaks (DSBs). Tungstate also modulated the activation of the central DSB signalling kinase, ATM, in response to these agents. These effects required the functionality of the Mre11–Nbs1–Rad50 (MRN) complex and were mimicked by the inhibition of PP2A phosphatase. Therefore, tungstate may have adjuvant activity when combined with DNA-damaging agents in the treatment of several malignancies.

Double-strand break repair by homologous recombination in primary mouse somatic cells requires BRCA1 but not the ATM kinase.

Homology-directed repair (HDR) is a critical pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. Efficient HDR is thought to be crucial for maintenance of genomic integrity during organismal development and tumor suppression. However, most mammalian HDR studies have focused on transformed and immortalized cell lines. We report here the generation of a Direct Repeat (DR)-GFP reporter-based mouse model to study HDR in primary cell types derived from diverse lineages. Embryonic and adult fibroblasts from these mice as well as cells derived from mammary epithelium, ovary, and neonatal brain were observed to undergo HDR at I-SceI endonuclease-induced DSBs at similar frequencies. When the DR-GFP reporter was crossed into mice carrying a hypomorphic mutation in the breast cancer susceptibility gene Brca1, a significant reduction in HDR was detected, showing that BRCA1 is critical for HDR in somatic cell types. Consistent with an HDR defect, Brca1 mutant mice are highly sensitive to the cross-linking agent mitomycin C. By contrast, loss of the DSB signaling ataxia telangiectasia-mutated (ATM) kinase did not significantly alter HDR levels, indicating that ATM is dispensable for HDR. Notably, chemical inhibition of ATM interfered with HDR. The DR-GFP mouse provides a powerful tool for dissecting the genetic requirements of HDR in a diverse array of somatic cell types in a normal, nontransformed cellular milieu.

SiRNA screening identifies differences in the Fanconi anemia pathway in BALB/c-Trp53+/− with susceptibility versus C57BL/6-Trp53+/− mice with resistance to mammary tumors

BALB/c mice heterozygous for Trp53 develop a high proportion of spontaneous mammary tumors, a phenotype distinct from other mouse strains. BALB/c-Trp53+/− female mice, thus, resemble the hereditary Li-Fraumeni syndrome (LFS) characterized by early-onset of breast cancer, even though LFS involves TP53 mutations, which may involve not only loss- but also gain-of-function. Previous analysis of tumors in BALB/c-Trp53+/− females showed frequent loss of heterozygosity involving the wild-type allele of Trp53 and displayed characteristics indicative of mitotic recombination. Critical involvement of DNA double-strand break (DSB) repair dysfunction, particularly of homologous recombination (HR), was also noticed in the etiology of human breast cancer. To better define functional alterations in BALB/c-Trp53+/− mice, we applied a fluorescence-based DSB repair assay on mouse embryonic fibroblasts (MEFs) from BALB/c-Trp53+/− versus C57BL/6J-Trp53+/− mice. This approach revealed deregulation of HR but not non-homologous end-joining (NHEJ) in BALB/c-Trp53+/−, which was further confirmed for mammary epithelial cells. Screening of a small interfering RNA-library targeting DSB repair, recombination, replication and signaling genes, identified 25 genes causing differences between homologous DSB repair in the two strains upon silencing. Interactome analysis of the hits revealed clustering of replication-related and fanconi anemia (FA)/breast cancer susceptibility (BRCA) genes. Further dissection of the functional change in BALB/c-Trp53+/− by immunofluorescence microscopy of nuclear 53BP1, Replication protein A (RPA) and Rad51 foci uncovered differences in crosslink and replication-associated repair. Chromosome breakage, G2 arrest and biochemical analyses indicated a FA pathway defect downstream of FancD2 associated with reduced levels of BRCA2. Consistent with polygenic models for BRCA, mammary carcinogenesis in BALB/c-Trp53+/− mice may, therefore, be promoted by a BRCA modifier allele in the FA pathway in the context of partial p53 loss-of-function.

Signaling of double strand breaks and deprotected telomeres in Arabidopsis.

Failure to repair DNA double strand breaks (DSB) can lead to chromosomal rearrangements and eventually to cancer or cell death. Radiation and environmental pollutants induce DSB and this is of particular relevance to plants due to their sessile life style. DSB also occur naturally in cells during DNA replication and programmed induction of DSB initiates the meiotic recombination essential for gametogenesis in most eukaryotes. The linear nature of most eukaryotic chromosomes means that each chromosome has two “broken” ends. Chromosome ends, or telomeres, are protected by nucleoprotein caps which avoid their recognition as DSB by the cellular DNA repair machinery. Deprotected telomeres are recognized as DSB and become substrates for recombination leading to chromosome fusions, the “bridge-breakage-fusion” cycle, genome rearrangements and cell death. The importance of repair of DSB and the severity of the consequences of their misrepair have led to the presence of multiple, robust mechanisms for their detection and repair. After a brief overview of DSB repair pathways to set the context, we present here an update of current understanding of the detection and signaling of DSB in the plant, Arabidopsis thaliana.

Systematic identification of functional residues in mammalian histone H2AX.

The histone variant H2AX is a principal component of chromatin involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). H2AX is thought to operate primarily through its C-terminal S139 phosphorylation, which mediates the recruitment of DNA damage response (DDR) factors to chromatin at DSB sites. Here, we describe a comprehensive screen of 67 residues in H2AX to determine their contributions to H2AX functions. Our analysis revealed that H2AX is both sumoylated and ubiquitylated. Individual residues defective for sumoylation, ubiquitylation, and S139 phosphorylation in untreated and damaged cells were identified. Specifically, we identified an acidic triad region in both H2A and H2AX that is required in cis for their ubiquitylation. We also report the characterization of a human H2AX knockout cell line, which exhibits DDR defects, including p53 activation, following DNA damage. Collectively, this work constitutes the first genetic complementation system for a histone in human cells. Finally, our data reveal new roles for several residues in H2AX and define distinct functions for H2AX in human cells.

Molecular Genetics of Breast and Ovarian Cancer: Recent Advances and Clinical Implications

Over the last few years, evidence has been accumulated that several susceptibility genes exist that differentially impact on the lifetime risk for breast or ovarian cancer. High-to-moderate penetrance alleles have been identified in genes involved in DNA double-strand break signaling and repair, and many low-penetrance susceptibility loci have been identified through genome-wide association studies. In this review, we briefly summarize present knowledge about breast and ovarian cancer susceptibility genes and discuss their implications for risk prediction and therapy.

ATM and 53BP1 Function in Nonoverlapping Pathways to Suppress T Lineage Lymphomagenesis.

Abstract 2374 Background:In response to DNA double-strand breaks (DSB), mammalian cells activate the DNA Damage Response (DDR), a network of factors that promotes their detection, signaling and repair. Among DDR factors, the Ataxia-Telangiectasia Mutated (ATM) kinase and the 53BP1 adaptor localize to chromatin surrounding DSBs, where 53BP1 is thought to transduce a subset of ATM-dependent reactions. In addition, 53BP1 may have ATM-independent functions via its interactions with the MRE11/RAD50/NBS1 (MRN) complex and others to protect DNA ends from degradation and to promote synapsis of distant DSBs. Both ATM and 53BP1 are tumor suppressors in humans and mice, but whether the combined mutation may aggravate the DSB repair defect and/or accelerate oncogenesis in vivo is currently unknown. Objectives:1) To generate a compound mouse model deficient for both ATM and 53BP1 and investigate their genetic interaction during growth and development; 2) To determine whether 53BP1 plays ATM-independent functions in DSB repair and cell cycle checkpoint activation in primary thymocytes; 3) To determine the effect of 53BP1 deficiency on the penetrance and latency of T cell lymphomas in ATM−/− mice. Design/Methods:We have generated 53BP1/ATM double knock out (DKO) mice and assessed the kinetics of DSB signaling and repair in primary thymocytes and mature B cells. We have also followed cohorts of DKO and control mice for tumor development and evaluated mechanisms of tumorigenesis using a combination of molecular, biochemical and cytogenetic approaches. Results:When compared to ATM−/− mice, DKO mice showed no differences in organismal growth and development, or in lymphoid differentiation. However, DKO mice developed T cell lymphomas with higher penetrance and at younger age than ATM−/− mice, and their survival was markedly reduced (p=0.0001; log-rank test). Like ATM−/− lymphomas, DKO lymphomas contained clonal translocations with breakpoints at the TCRα/δ locus and amplification of upstream DNA sequences, indicating that 53BP1 deficiency markedly accelerates the acquisition of oncogenic translocations in an ATM−/− background. In primary cells, loss of 53BP1 further increases the frequency of ionizing irradiation (IR)-induced chromosomal aberrations in ATM−/− cells. We will present a model for how 53BP1 actions at DSBs suppress genomic instability in ATM-deficient thymocytes, including its effects on checkpoint activation and DSB repair. Conclusion:Our studies in a mouse model of T cell lymphomagenesis suggest that mutations in 53BP1 and ATM may be nonmutually exclusive in human lymphomas. The combined mutation may define a subgroup of patients with more aggressive hematological malignancies, and may have implications for their response to therapy with DNA damaging agents. Disclosures:No relevant conflicts of interest to declare.

High Activity of Poly(ADP-Ribose) Polymerase in Chronic Lymphocytic Leukemia

AbstractAbstract 1788Current treatment protocols for Chronic lymphocytic leukemia (CLL), including FCR (fludarabine, cyclophosphamide and rituximab) have improved response rates and newer approaches targeting the B-cell receptor show promise in early clinical trials. Despite these advances, CLL remains incurable, and since CLL patients are predominantly elderly, many cannot tolerate more aggressive therapies. Therefore, development of biomarkers to stratify subgroups of patients for personalised medicine is paramount to achieve the best therapeutic outcomes for these patients.Chemo-resistance in CLL frequently occurs due to loss of function of either p53 or ataxia telangiectasia mutated kinase (ATM) (del(17p) and del(11q) respectively). p53 and ATM mutation and/or deletion results in defective DNA double-strand break (DSB) response pathways, which confer greater genomic instability, poorer response and shorter overall survival (OS). ATM is a key DSB signaling kinase with roles in homologous recombination repair and cell cycle checkpoint activation, which facilitate DNA repair. We hypothesized that as well as defects in DSB-activated enzymes, genomic instability arising from unresolved single-strand DNA breaks may play a role in disease progression and resistance in CLL.Poly (ADP-ribose) polymerase (PARP) is activated by DNA damaging agents that induce single strand breaks (e.g. alkylating agents) and is pivotal in the signaling to direct repair of such DNA damage. Therefore, inhibitors of PARP sensitise cells to certain classes of chemotherapeutic agents by inhibiting DNA repair. PARP is also the first line of defence against oxidative stress, which is associated with poor prognosis in CLL. We developed the first clinically-used PARP inhibitor (PARPi) and subsequent studies identified the synthetic lethality of PARPi monotherapy in homologous recombination repair defective (HRD) cancer, which selectively kills HRD tumour cells, but is non-toxic to the HR-competent normal tissues. Importantly, PARP activity is reported to be higher in HRD cells, which may reflect increased genomic instability and oxidative stress in these cells. Currently, there are several PARPi undergoing clinical evaluation, including two studies in CLL patients.Here, we analysed the activity of PARP in CLL, to determine whether it could be used as a biomarker of response to conventional therapy and a means to stratify patients for novel therapies, including PARP inhibitor (PARPi) monotherapy. Using the clinically validated PARP activity assay, we found that PARP activity in PBMCs from healthy volunteers was in the same range as we had previously observed (Zaremba T, Biochem J 2011). However when we measured PARP activity in CLL cells (n=33 cases) it was up to 100-fold higher than the activity in PBMCs from healthy volunteers. Activity ranged from 100-50, 000 pmoL/106 cells and was higher in those patients with Binet stage C, or progressive disease. When patients were stratified according to ATM functional status (determined by measuring ATM activity) PARP activity was significantly higher in cases with confirmed ATM dysfunction (p=0.02), compared to those with functional ATM. We then designated cases as having PARP activity that was either higher than, or lower than the median (12,400 pmoL/106cells) and performed Kaplan Meier analysis. There was a trend towards high PARP activity being associated with shorter OS.These results could have significant ramifications: recent reports support the concept that ATM-defective CLL can call be selectively targeted by PARPi (Weston VJ, Blood 2010) and therefore current phase I clinical trials are investigating the use of PARPi in HRD (including ATM-defective) CLL.These trials are stratifying patients by del(11q) and ATM status (since ATM plays a key role in mediating successful HR) with the aim of selectively killing ATM-defective CLL cells with the PARPi. However, ATM status alone may not be sufficient for patient stratification. We propose that CLL cells with high PARP activity are “addicted” to PARP by virtue of its role protecting against increased DNA damage, and that PARP activity may be a key determinant of patient sensitivity to PARP inhibitors. Additionally, the underlying mechanism for high PARP activity in CLL requires further investigation, since this may reveal new therapeutic options, and strategies for patient stratification prior to clinical trial. Disclosures:No relevant conflicts of interest to declare.

Cleavage of the BRCT tandem domains of nibrin by the 657del5 mutation affects the DNA damage response less than the Arg215Trp mutation

The Nijmegen breakage syndrome (NBS) is a genetic disorder caused by mutations in NBN gene and characterized by chromosomal instability and hypersensitivity to ionizing radiations (IR). The N-terminus of nibrin (NBN) contains a tandem breast cancer 1 (BRCA1) carboxy-terminal (BRCT) domain that represents one of the major mediators of phosphorylation-dependent protein-protein interactions in processes related to cell cycle checkpoint and DNA repair functions. Patients with NBS compound heterozygous for the 657del5 hypomorphic mutation and for the Arg215Trp missense mutation (corresponding to the 643C>T gene mutation) display a clinical phenotype more severe than that of patients homozygous for the 657del5 mutation. Here, we show that both the 657del5 and Arg215Trp mutations, occurring within the tandem BRCT domains of NBN, although not altering the assembly of the MRE11/RAD50/NBN (MRN) complex, affect the MRE11 IR-induced nuclear foci (IRIF) formation and the DNA double-strand break (DSB) signaling via the phosphorylation of both ataxia-telangiectasia-mutated (ATM) kinase and ATM downstream targets (e.g., SMC1 and p53). Remarkably, data obtained indicate that the cleavage of the BRCT tandem domains of NBN by the 657del5 mutation affects the DNA damage response less than the Arg215Trp mutation. Indeed, the 70-kDa NBN fragment, arising from the 657del5 mutation, maintains the capability to interact with MRE11 and γ-H2AX and to form IRIF. Altogether, the role of the tandem BRCT domains of NBN in the localization of the MRN complex at the DNA DSB and in the activation of the damage response is highlighted.

Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography

The faithful maintenance of chromosome continuity in human cells during DNA replication and repair is critical for preventing the conversion of normal diploid cells to an oncogenic state. The evolution of higher eukaryotic cells endowed them with a large genetic investment in the molecular machinery that ensures chromosome stability. In mammalian and other vertebrate cells, the elimination of double-strand breaks with minimal nucleotide sequence change involves the spatiotemporal orchestration of a seemingly endless number of proteins ranging in their action from the nucleotide level to nucleosome organization and chromosome architecture. DNA DSBs trigger a myriad of post-translational modifications that alter catalytic activities and the specificity of protein interactions: phosphorylation, acetylation, methylation, ubiquitylation, and SUMOylation, followed by the reversal of these changes as repair is completed. "Superfluous" protein recruitment to damage sites, functional redundancy, and alternative pathways ensure that DSB repair is extremely efficient, both quantitatively and qualitatively. This review strives to integrate the information about the molecular mechanisms of DSB repair that has emerged over the last two decades with a focus on DSBs produced by the prototype agent ionizing radiation (IR). The exponential growth of molecular studies, heavily driven by RNA knockdown technology, now reveals an outline of how many key protein players in genome stability and cancer biology perform their interwoven tasks, e.g. ATM, ATR, DNA-PK, Chk1, Chk2, PARP1/2/3, 53BP1, BRCA1, BRCA2, BLM, RAD51, and the MRE11-RAD50-NBS1 complex. Thus, the nature of the intricate coordination of repair processes with cell cycle progression is becoming apparent. This review also links molecular abnormalities to cellular pathology as much a possible and provides a framework of temporal relationships.