PBAF loss leads to DNA damage-induced inflammatory signaling through defective G2/M checkpoint maintenance.

The PBRM1 subunit of the PBAF (SWI/SNF) chromatin remodeling complex is mutated in ∼40% of clear cell renal cancers. PBRM1 loss has been implicated in responses to immunotherapy in renal cancer, but the mechanism is unclear. DNA damage-induced inflammatory signaling is an important factor determining immunotherapy response. This response is kept in check by the G2/M checkpoint, which prevents progression through mitosis with unrepaired damage. We found that in the absence of PBRM1, p53-dependent p21 up-regulation is delayed after DNA damage, leading to defective transcriptional repression by the DREAM complex and premature entry into mitosis. Consequently, DNA damage-induced inflammatory signaling pathways are activated by cytosolic DNA. Notably, p53 is infrequently mutated in renal cancer, so PBRM1 mutational status is critical to G2/M checkpoint maintenance. Moreover, we found that the ability of PBRM1 deficiency to predict response to immunotherapy correlates with expression of the cytosolic DNA-sensing pathway in clinical samples. These findings have implications for therapeutic responses in renal cancer.

Repression of Transcription at DNA Breaks Requires Cohesin throughout Interphase and Prevents Genome Instability.

Cohesin subunits are frequently mutated in cancer, but how they function as tumor suppressors is unknown. Cohesin mediates sister chromatid cohesion, but this is not always perturbed in cancer cells. Here, we identify a previously unknown role for cohesin. We find that cohesin is required to repress transcription at DNA double-strand breaks (DSBs). Notably, cohesin represses transcription at DSBs throughout interphase, indicating that this is distinct from its known role in mediating DNA repair through sister chromatid cohesion. We identified a cancer-associated SA2 mutation that supports sister chromatid cohesion but is unable to repress transcription at DSBs. We further show that failure to repress transcription at DSBs leads to large-scale genome rearrangements. Cancer samples lacking SA2 display mutational patterns consistent with loss of this pathway. These findings uncover a new function for cohesin that provides insights into its frequent loss in cancer.

DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination.

Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis.

Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin.

Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity.

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.

A Process of Resection-Dependent Nonhomologous End Joining Involving the Goddess Artemis.

DNA double-strand breaks (DSBs) are a hazardous form of damage that can potentially cause cell death or genomic rearrangements. In mammalian G1- and G2-phase cells, DSBs are repaired with two-component kinetics. In both phases, a fast process uses canonical nonhomologous end joining (c-NHEJ) to repair the majority of DSBs. In G2, slow repair occurs by homologous recombination. The slow repair process in G1 also involves c-NHEJ proteins but additionally requires the nuclease Artemis and DNA end resection. Here, we consider the nature of slow DSB repair in G1 and evaluate factors determining whether DSBs are repaired with fast or slow kinetics. We consider limitations in our current knowledge and present a speculative model for Artemis-dependent c-NHEJ and the environment underlying its usage.

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

A coordinated DNA damage response promotes adult quiescent neural stem cell activation.

Stem and differentiated cells frequently differ in their response to DNA damage, which can determine tissue sensitivity. By exploiting insight into the spatial arrangement of subdomains within the adult neural subventricular zone (SVZ) in vivo, we show distinct responses to ionising radiation (IR) between neural stem and progenitor cells. Further, we reveal different DNA damage responses between neonatal and adult neural stem cells (NSCs). Neural progenitors (transit amplifying cells and neuroblasts) but not NSCs (quiescent and activated) undergo apoptosis after 2 Gy IR. This response is cell type- rather than proliferation-dependent and does not appear to be driven by distinctions in DNA damage induction or repair capacity. Moreover, exposure to 2 Gy IR promotes proliferation arrest and differentiation in the adult SVZ. These 3 responses are ataxia telangiectasia mutated (ATM)-dependent and promote quiescent NSC (qNSC) activation, which does not occur in the subdomains that lack progenitors. Neuroblasts arising post-IR derive from activated qNSCs rather than irradiated progenitors, minimising damage compounded by replication or mitosis. We propose that rather than conferring sensitive cell death, apoptosis is a form of rapid cell death that serves to remove damaged progenitors and promote qNSC activation. Significantly, analysis of the neonatal (P5) SVZ reveals that although progenitors remain sensitive to apoptosis, they fail to efficiently arrest proliferation. Consequently, their repopulation occurs rapidly from irradiated progenitors rather than via qNSC activation.

Regulation of programmed death-ligand 1 expression in response to DNA damage in cancer cells: Implications for precision medicine.

Anti-programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) therapy, which is one of the most promising cancer therapies, is licensed for treating various tumors. Programmed death-ligand 1, which is expressed on the surface of cancer cells, leads to the inhibition of T lymphocyte activation and immune evasion if it binds to the receptor PD-1 on CTLs. Anti-PD-1/PD-L1 Abs inhibit interactions between PD-1 and PD-L1 to restore antitumor immunity. Although certain patients achieve effective responses to anti-PD-1/PD-L1 therapy, the efficacy of treatment is highly variable. Clinical trials of anti-PD-1/PD-L1 therapy combined with radiotherapy/chemotherapy are underway with suggestive evidence of favorable outcome; however, the molecular mechanism is largely unknown. Among several molecular targets that can influence the efficacy of anti-PD-1/PD-L1 therapy, PD-L1 expression in tumors is considered to be a critical biomarker because there is a positive correlation between the efficacy of combined treatment protocols and PD-L1 expression levels. Therefore, understanding the mechanisms underlying the regulation of PD-L1 expression in cancer cells, particularly the mechanism of PD-L1 expression following DNA damage, is important. In this review, we consider recent findings on the regulation of PD-L1 expression in response to DNA damage signaling in cancer cells.

The tubercular badger and the uncertain curve:- The need for a multiple stressor approach in environmental radiation protection.

This article presents the results of a workshop held in Stirling, Scotland in June 2018, called to examine critically the effects of low-dose ionising radiation on the ecosphere. The meeting brought together participants from the fields of low- and high-dose radiobiology and those working in radioecology to discuss the effects that low doses of radiation have on non-human biota. In particular, the shape of the low-dose response relationship and the extent to which the effects of low-dose and chronic exposure may be predicted from high dose rate exposures were discussed. It was concluded that high dose effects were not predictive of low dose effects. It followed that the tools presently available were deemed insufficient to reliably predict risk of low dose exposures in ecosystems. The workshop participants agreed on three major recommendations for a path forward. First, as treating radiation as a single or unique stressor was considered insufficient, the development of a multidisciplinary approach is suggested to address key concerns about multiple stressors in the ecosphere. Second, agreed definitions are needed to deal with the multiplicity of factors determining outcome to low dose exposures as a term can have different meanings in different disciplines. Third, appropriate tools need to be developed to deal with the different time, space and organisation level scales. These recommendations permit a more accurate picture of prospective risks.

ATR promotes cilia signalling: links to developmental impacts.

Mutations in ATR(ataxia telangiectasia and RAD3-related) cause Seckel syndrome (ATR-SS), a microcephalic primordial dwarfism disorder. Hitherto, the clinical manifestation of ATR deficiency has been attributed to its canonical role in DNA damage response signalling following replication fork stalling/collapse. Here, we show that ATR regulates cilia-dependent signalling in a manner that can be uncoupled from its function during replication. ATR-depleted or patient-derived ATR-SS cells form cilia of slightly reduced length but are dramatically impaired in cilia-dependent signalling functions, including growth factor and Sonic hedgehog signalling. To better understand the developmental impact of ATR loss of function, we also used zebrafish as a model. Zebrafish embryos depleted of Atr resembled ATR-SS morphology, showed a modest but statistically significant reduction in cilia length and other morphological features indicative of cilia dysfunction. Additionally, they displayed defects in left-right asymmetry including ambiguous expression of southpaw, incorrectly looped hearts and randomized localization of internal organs including the pancreas, features typically conferred by cilia dysfunction. Our findings reveal a novel role for ATR in cilia signalling distinct from its canonical function during replication and strengthen emerging links between cilia function and development.

Low levels of endogenous or X-ray-induced DNA double-strand breaks activate apoptosis in adult neural stem cells.

The embryonic neural stem cell compartment is characterised by rapid proliferation from embryonic day (E)11 to E16.5, high endogenous DNA double-strand break (DSB) formation and sensitive activation of apoptosis. Here, we ask whether DSBs arise in the adult neural stem cell compartments, the sub-ventricular zone (SVZ) of the lateral ventricles and the sub-granular zone (SGZ) of the hippocampal dentate gyrus, and whether they activate apoptosis. We used mice with a hypomorphic mutation in DNA ligase IV (Lig4(Y288C)), ataxia telangiectasia mutated (Atm(-/-)) and double mutant Atm(-/-)/Lig4(Y288C) mice. We demonstrate that, although DSBs do not arise at a high frequency in adult neural stem cells, the low numbers of DSBs that persist endogenously in Lig4(Y288C) mice or that are induced by low radiation doses can activate apoptosis. A temporal analysis shows that DSB levels in Lig4(Y288C) mice diminish gradually from the embryo to a steady state level in adult mice. The neonatal SVZ compartment of Lig4(Y288C) mice harbours diminished DSBs compared to its differentiated counterpart, suggesting a process selecting against unfit stem cells. Finally, we reveal high endogenous apoptosis in the developing SVZ of wild-type newborn mice.

A restatement of the natural science evidence base concerning the health effects of low-level ionizing radiation.

Exposure to ionizing radiation is ubiquitous, and it is well established that moderate and high doses cause ill-health and can be lethal. The health effects of low doses or low dose-rates of ionizing radiation are not so clear. This paper describes a project which sets out to summarize, as a restatement, the natural science evidence base concerning the human health effects of exposure to low-level ionizing radiation. A novel feature, compared to other reviews, is that a series of statements are listed and categorized according to the nature and strength of the evidence that underpins them. The purpose of this restatement is to provide a concise entrée into this vibrant field, pointing the interested reader deeper into the literature when more detail is needed. It is not our purpose to reach conclusions on whether the legal limits on radiation exposures are too high, too low or just right. Our aim is to provide an introduction so that non-specialist individuals in this area (be they policy-makers, disputers of policy, health professionals or students) have a straightforward place to start. The summary restatement of the evidence and an extensively annotated bibliography are provided as appendices in the electronic supplementary material.

SETDB1, HP1 and SUV39 promote repositioning of 53BP1 to extend resection during homologous recombination in G2 cells.

Recent studies have shown that homologous recombination (HR) requires chromatin repression as well as relaxation at DNA double strand breaks (DSBs). HP1 and SUV39H1/2 are repressive factors essential for HR. Here, we identify SETDB1 as an additional compacting factor promoting HR. Depletion of HP1, SUV39, SETDB1 or BRCA1 confer identical phenotypes. The repressive factors, like BRCA1, are dispensable for the initiation of resection but promote the extension step causing diminished RPA or RAD51 foci and HR in irradiated G2 cells. Depletion of the compacting factors does not inhibit BRCA1 recruitment but at 8 h post IR, BRCA1 foci are smaller and aberrantly positioned compared to control cells. BRCA1 promotes 53BP1 repositioning to the periphery of enlarged foci and formation of a devoid core with BRCA1 becoming enlarged and localized internally to 53BP1. Depletion of the compacting factors precludes these changes at irradiation-induced foci. Thus, the repressive factors are required for BRCA1 function in promoting the repositioning of 53BP1 during HR. Additionally, depletion of these repressive factors in undamaged cells causes diminished sister chromatid association at centromeric sequences. We propose a model for how these findings may be functionally linked.

ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin.

Ataxia Telangiectasia Mutated (ATM) signaling is essential for the repair of a subset of DNA double-strand breaks (DSBs); however, its precise role is unclear. Here, we show that < or =25% of DSBs require ATM signaling for repair, and this percentage correlates with increased chromatin but not damage complexity. Importantly, we demonstrate that heterochromatic DSBs are generally repaired more slowly than euchromatic DSBs, and ATM signaling is specifically required for DSB repair within heterochromatin. Significantly, knockdown of the transcriptional repressor KAP-1, an ATM substrate, or the heterochromatin-building factors HP1 or HDAC1/2 alleviates the requirement for ATM in DSB repair. We propose that ATM signaling temporarily perturbs heterochromatin via KAP-1, which is critical for DSB repair/processing within otherwise compacted/inflexible chromatin. In support of this, ATM signaling alters KAP-1 affinity for chromatin enriched for heterochromatic factors. These data suggest that the importance of ATM signaling for DSB repair increases as the heterochromatic component of a genome expands.

How cancer cells hijack DNA double-strand break repair pathways to gain genomic instability.

DNA DSBs (double-strand breaks) are a significant threat to the viability of a normal cell, since they can result in loss of genetic material if mitosis or replication is attempted in their presence. Consequently, evolutionary pressure has resulted in multiple pathways and responses to enable DSBs to be repaired efficiently and faithfully. Cancer cells, which are under pressure to gain genomic instability, have a striking ability to avoid the elegant mechanisms by which normal cells maintain genomic stability. Current models suggest that, in normal cells, DSB repair occurs in a hierarchical manner that promotes rapid and efficient rejoining first, with the utilization of additional steps or pathways of diminished accuracy if rejoining is unsuccessful or delayed. In the present review, we evaluate the fidelity of DSB repair pathways and discuss how cancer cells promote the utilization of less accurate processes. Homologous recombination serves to promote accuracy and stability during replication, providing a battlefield for cancer to gain instability. Non-homologous end-joining, a major DSB repair pathway in mammalian cells, usually operates with high fidelity and only switches to less faithful modes if timely repair fails. The transition step is finely tuned and provides another point of attack during tumour progression. In addition to DSB repair, a DSB signalling response activates processes such as cell cycle checkpoint arrest, which enhance the possibility of accurate DSB repair. We consider the ways by which cancers modify and hijack these processes to gain genomic instability.

Deficiency in origin licensing proteins impairs cilia formation: implications for the aetiology of Meier-Gorlin syndrome.

Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA-mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency.

XRCC4 deficiency in human subjects causes a marked neurological phenotype but no overt immunodeficiency.

BACKGROUND: Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair mechanism in human cells. The final rejoining step requires DNA ligase IV (LIG4) together with the partner proteins X-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor. Patients with mutations in genes encoding LIG4, XRCC4-like factor, or the other NHEJ proteins DNA-dependent protein kinase catalytic subunit and Artemis are DSB repair defective and immunodeficient because of the requirement for NHEJ during V(D)J recombination. OBJECTIVE: We found a patient displaying microcephaly and progressive ataxia but a normal immune response. We sought to determine pathogenic mutations and to describe the molecular pathogenesis of the patient. METHODS: We performed next-generation exome sequencing. We evaluated the DSB repair activities and V(D)J recombination capacity of the patient's cells, as well as performing a standard blood immunologic characterization. RESULTS: We identified causal mutations in the XRCC4 gene. The patient's cells are radiosensitive and display the most severe DSB repair defect we have encountered using patient-derived cell lines. In marked contrast, a V(D)J recombination plasmid assay revealed that the patient's cells did not display the junction abnormalities that are characteristic of other NHEJ-defective cell lines. The mutant protein can interact efficiently with LIG4 and functions normally in in vitro assays and when transiently expressed in vivo. However, the mutation makes the protein unstable, and it undergoes proteasome-mediated degradation. CONCLUSION: Our findings reveal a novel separation of impact phenotype: there is a pronounced DSB repair defect and marked clinical neurological manifestation but no clinical immunodeficiency.

Factors determining DNA double-strand break repair pathway choice in G2 phase.

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.