A Genetic Map of the Response to DNA Damage in Human Cells.

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

Transcription recovery after DNA damage requires chromatin priming by the H3.3 histone chaperone HIRA.

Understanding how to recover fully functional and transcriptionally active chromatin when its integrity has been challenged by genotoxic stress is a critical issue. Here, by investigating how chromatin dynamics regulate transcriptional activity in response to DNA damage in human cells, we identify a pathway involving the histone chaperone histone regulator A (HIRA) to promote transcription restart after UVC damage. Our mechanistic studies reveal that HIRA accumulates at sites of UVC irradiation upon detection of DNA damage prior to repair and deposits newly synthesized H3.3 histones. This local action of HIRA depends on ubiquitylation events associated with damage recognition. Furthermore, we demonstrate that the early and transient function of HIRA in response to DNA damage primes chromatin for later reactivation of transcription. We propose that HIRA-dependent histone deposition serves as a chromatin bookmarking system to facilitate transcription recovery after genotoxic stress.

Endogenous DNA 3' Blocks Are Vulnerabilities for BRCA1 and BRCA2 Deficiency and Are Reversed by the APE2 Nuclease.

The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.

BRCA1 Haploinsufficiency Is Masked by RNF168-Mediated Chromatin Ubiquitylation.

BRCA1 functions at two distinct steps during homologous recombination (HR). Initially, it promotes DNA end resection, and subsequently it recruits the PALB2 and BRCA2 mediator complex, which stabilizes RAD51-DNA nucleoprotein filaments. Loss of 53BP1 rescues the HR defect in BRCA1-deficient cells by increasing resection, suggesting that BRCA1's downstream role in RAD51 loading is dispensable when 53BP1 is absent. Here we show that the E3 ubiquitin ligase RNF168, in addition to its canonical role in inhibiting end resection, acts in a redundant manner with BRCA1 to load PALB2 onto damaged DNA. Loss of RNF168 negates the synthetic rescue of BRCA1 deficiency by 53BP1 deletion, and it predisposes BRCA1 heterozygous mice to cancer. BRCA1+/-RNF168-/- cells lack RAD51 foci and are hypersensitive to PARP inhibitor, whereas forced targeting of PALB2 to DNA breaks in mutant cells circumvents BRCA1 haploinsufficiency. Inhibiting the chromatin ubiquitin pathway may, therefore, be a synthetic lethality strategy for BRCA1-deficient cancers.

Control of homologous recombination by the HROB-MCM8-MCM9 pathway.

DNA repair by homologous recombination (HR) is essential for genomic integrity, tumor suppression, and the formation of gametes. HR uses DNA synthesis to repair lesions such as DNA double-strand breaks and stalled DNA replication forks, but despite having a good understanding of the steps leading to homology search and strand invasion, we know much less of the mechanisms that establish recombination-associated DNA polymerization. Here, we report that C17orf53/HROB is an OB-fold-containing factor involved in HR that acts by recruiting the MCM8-MCM9 helicase to sites of DNA damage to promote DNA synthesis. Mice with targeted mutations in Hrob are infertile due to depletion of germ cells and display phenotypes consistent with a prophase I meiotic arrest. The HROB-MCM8-MCM9 pathway acts redundantly with the HELQ helicase, and cells lacking both HROB and HELQ have severely impaired HR, suggesting that they underpin two major routes for the completion of HR downstream from RAD51. The function of HROB in HR is reminiscent of that of gp59, which acts as the replicative helicase loader during bacteriophage T4 recombination-dependent DNA replication. We therefore propose that the loading of MCM8-MCM9 by HROB may similarly be a key step in the establishment of mammalian recombination-associated DNA synthesis.

The Histone Chaperone FACT Coordinates H2A.X-Dependent Signaling and Repair of DNA Damage.

Safeguarding cell function and identity following a genotoxic stress challenge entails a tight coordination of DNA damage signaling and repair with chromatin maintenance. How this coordination is achieved and with what impact on chromatin integrity remains elusive. Here, we address these questions by investigating the mechanisms governing the distribution in mammalian chromatin of the histone variant H2A.X, a central player in damage signaling. We reveal that H2A.X is deposited de novo at sites of DNA damage in a repair-coupled manner, whereas the H2A.Z variant is evicted, thus reshaping the chromatin landscape at repair sites. Our mechanistic studies further identify the histone chaperone FACT (facilitates chromatin transcription) as responsible for the deposition of newly synthesized H2A.X. Functionally, we demonstrate that FACT potentiates H2A.X-dependent signaling of DNA damage. We propose that new H2A.X deposition in chromatin reflects DNA damage experience and may help tailor DNA damage signaling to repair progression.

The shieldin complex mediates 53BP1-dependent DNA repair.

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14-9, the latter of which recruits REV7 (also known as MAD2L2) to break sites10,11. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

Real-Time Tracking of Parental Histones Reveals Their Contribution to Chromatin Integrity Following DNA Damage.

Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage.

Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites.

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin-dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80- and RING-dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding-deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80-BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.

Identifying classes of the pain, fatigue, and depression symptom cluster in long-term prostate cancer survivors-results from the multi-regional Prostate Cancer Survivorship Study in Switzerland (PROCAS).

PURPOSE: Aside from urological and sexual problems, long-term (≥5 years after initial diagnosis) prostate cancer (PC) survivors might suffer from pain, fatigue, and depression. These concurrent symptoms can form a cluster. In this study, we aimed to investigate classes of this symptom cluster in long-term PC survivors, to classify PC survivors accordingly, and to explore associations between classes of this cluster and health-related quality of life (HRQoL). METHODS: Six hundred fifty-three stage T1-T3N0M0 survivors were identified from the Prostate Cancer Survivorship in Switzerland (PROCAS) study. Fatigue was assessed with the EORTC QLQ-FA12, depressive symptoms with the MHI-5, and pain with the EORTC QLQ-C30 questionnaire. Latent class analysis was used to derive cluster classes. Factors associated with the derived classes were determined using multinomial logistic regression analysis. RESULTS: Three classes were identified: class 1 (61.4%) - "low pain, low physical and emotional fatigue, moderate depressive symptoms"; class 2 (15.1%) - "low physical fatigue and pain, moderate emotional fatigue, high depressive symptoms"; class 3 (23.5%) - high scores for all symptoms. Survivors in classes 2 and 3 were more likely to be physically inactive, report a history of depression or some other specific comorbidity, be treated with radiation therapy, and have worse HRQoL outcomes compared to class 1. CONCLUSION: Three distinct classes of the pain, fatigue, and depression cluster were identified, which are associated with treatment, comorbidities, lifestyle factors, and HRQoL outcomes. Improving classification of PC survivors according to severity of multiple symptoms could assist in developing interventions tailored to survivors' needs.

Age-specific health-related quality of life in disease-free long-term prostate cancer survivors versus male population controls-results from a population-based study.

BACKGROUND: Prostate cancer (PC) and its treatment may affect PC survivors differently with respect to age. However, little is known regarding age-specific health-related quality of life (HRQoL) in PC survivors 5 years or even ≥ 10 years post-diagnosis. METHODS: The sample included 1975 disease-free PC survivors (5-16 years post-diagnosis) and 661 cancer-free population controls, recruited from two German population-based studies (CAESAR+, LinDe). HRQoL in both populations was assessed using the EORTC QLQ-C30 questionnaire. Additionally, PC survivors completed the PC-specific EORTC QLQ-PR25 questionnaire. Differences in HRQoL between survivors and controls, as well as differences according to age and time since diagnosis were analyzed with multiple regression after adjustment for age, education, stage, and time since diagnosis, where appropriate. RESULTS: In general, PC survivors reported HRQoL and symptom-burden levels comparable to the general population, except for significantly poorer social functioning and higher burden for diarrhea and constipation. In age-specific analyses, PC survivors up to 69 years indicated poorer global health and social functioning than population controls. Stratification by time since diagnosis revealed little difference between the subgroups. On PC-specific symptoms, burden was highest for urinary bother and symptoms, and lowest for bowel symptoms. Younger age was associated with less urinary symptoms but higher urinary bother. CONCLUSION: Long-term disease-free PC survivors reported overall good HRQoL, but experienced persistent specific detriments. Our data suggest that these detriments do not improve substantially with increasing time since diagnosis. Targeted interventions are recommended to prevent PC-related and treatment-related symptoms becoming chronic and to enhance social functioning.

Health-related quality of life in long-term survivors with localised prostate cancer by therapy-Results from a population-based study.

OBJECTIVE: Several therapies for localised prostate cancer (PC) are available; all yield similar survival rates. However, each therapy has significant side effects that can influence patients' health-related quality of life (HRQoL) in the long run. METHODS: The study sample included 911 survivors with localised PC, 5-15 years post-diagnosis who were identified from the population-based CAESAR + study in Germany. HRQoL was assessed using the EORTC QLQ-C30 and EORTC QLQ-PR25 questionnaires. The association between type of therapy and HRQoL was assessed with multivariable linear regression and global F-test adjusting for age, time since diagnosis and comorbidities. RESULTS: Overall, survivors treated with radical prostatectomy (RP) or radiotherapy (RT) alone reported the best HRQoL and the lowest symptom burden. Conversely, survivors treated with androgen deprivation therapy (ADT) (& RP/RT) or RP & RT (in combination) reported the worst HRQoL and the highest symptom burden. Significant differences among treatment groups in HRQoL were found for global health status (p = 0.041), social functioning (p = 0.007), urinary symptoms (p = 0.035), bowel symptoms (p = 0.017) and hormonal treatment-related symptoms (p < 0.001) among other symptoms. CONCLUSIONS: Long-term localised PC survivors formerly treated with a combination of RP and RT or with ADT report poorer HRQoL and more symptoms than patients treated with either RP or RT alone.

Correction to: Health-related quality of life among long-term (≥5 years) prostate cancer survivors by primary intervention: a systematic review.

The original article [1] contains errors whereby some information provided in Tables 2 and 5 in the online version is missing in the PDF version; in addition, some details regarding the study by Mols et al., Johnstone et al. and Fransson et al. (2008) in Tables 1 and 5 require correction.

Health-related quality of life among long-term (≥5 years) prostate cancer survivors by primary intervention: a systematic review.

BACKGROUND: Due to an improving prognosis, and increased knowledge of intervention effects over time, long-term well-being among prostate cancer (PC) survivors has gained increasing attention. Yet, despite a variety of available PC interventions, experts currently disagree on optimal intervention course based on survival rates. METHODS: In January 2017, we searched multiple databases to identify relevant articles. Studies were required to assess at least two different dimensions of health-related quality of life (HRQoL) in PC survivors ≥5 years past diagnosis with validated measures. RESULTS: Identified studies (n = 13) were mainly observational cohort studies (n = 10), conducted in developed countries with a sample size below 100 per study arm (n = 6). External-beam radiation therapy was the most common intervention (n = 12), whereas only three studies included patients on active surveillance or on watchful waiting. Studies were largely heterogeneous in cancer stage at diagnosis, intervention groups and instruments. All identified studies either used the EORTC QLQ-C30 (n = 5) or the SF-36 (n = 7) to assess generic HRQoL, yet 11 different instruments were employed to assess PC specific urinary, bowel and sexual symptoms. Overall, no consistent pattern between intervention and HRQoL was observed. Results from two randomized-controlled-trials (RCTs) and one observational study, comparing HRQoL by primary intervention in localized PC survivors suggest that long-term HRQoL does not differ by intervention. However, observational studies that included a combination of localized and locally advanced stage PC survivors identified HRQoL differences for various scales including physical well-being, social and role function, vitality, and role emotional. CONCLUSION: This review reveals the number of publications comparing HRQoL by primary intervention in long-term PC survivors is currently limited. Robust data from two RCTs and one observational study suggest that HRQoL does not seem to differ by intervention. However, the heterogeneity of studies' methodologies and results hindered our ability to draw a clear conclusion. Therefore, in order to answer the question of which primary intervention is superior with respect to long-term HRQoL in PC patients, more high-quality, large-scale prospective cohort studies, or RCTs with repeated HRQoL assessments, are urgently needed.

Blurring the line between the DNA damage response and transcription: the importance of chromatin dynamics.

DNA damage interferes with the progression of transcription machineries. A tight coordination of transcription with signaling and repair of DNA damage is thus critical for safeguarding genome function. This coordination involves modulations of chromatin organization. Here, we focus on the central role of chromatin dynamics, in conjunction with DNA Damage Response (DDR) factors, in controlling transcription inhibition and restart at sites of DNA damage in mammalian cells. Recent work has identified chromatin modifiers and histone chaperones as key regulators of transcriptional activity in damaged chromatin regions. Conversely, the transcriptional state of chromatin before DNA damage influences both DNA damage signaling and repair. We discuss the importance of chromatin plasticity in coordinating the interplay between the DDR and transcription, with major implications for cell fate maintenance.

CTNNBL1 is a novel nuclear localization sequence-binding protein that recognizes RNA-splicing factors CDC5L and Prp31.

Nuclear proteins typically contain short stretches of basic amino acids (nuclear localization sequences; NLSs) that bind karyopherin α family members, directing nuclear import. Here, we identify CTNNBL1 (catenin-ß-like 1), an armadillo motif-containing nuclear protein that exhibits no detectable primary sequence homology to karyopherin α, as a novel, selective NLS-binding protein. CTNNBL1 (a single-copy gene conserved from fission yeast to man) was previously found associated with Prp19-containing RNA-splicing complexes as well as with the antibody-diversifying enzyme AID. We find that CTNNBL1 association with the Prp19 complex is mediated by recognition of the NLS of the CDC5L component of the complex and show that CTNNBL1 also interacts with Prp31 (another U4/U6.U5 tri-snRNP-associated splicing factor) through its NLS. As with karyopherin αs, CTNNBL1 binds NLSs via its armadillo (ARM) domain, but displays a separate, more selective NLS binding specificity. Furthermore, the CTNNBL1/AID interaction depends on amino acids forming the AID conformational NLS with CTNNBL1-deficient cells showing a partial defect in AID nuclear accumulation. However, in further contrast to karyopherin αs, the CTNNBL1 N-terminal region itself binds karyopherin αs (rather than karyopherin ß), suggesting a function divergent from canonical nuclear transport. Thus, CTNNBL1 is a novel NLS-binding protein, distinct from karyopherin αs, with the results suggesting a possible role in the selective intranuclear targeting or interactions of some splicing-associated complexes.

Chromatin dynamics during nucleotide excision repair: histones on the move.

It has been a long-standing question how DNA damage repair proceeds in a nuclear environment where DNA is packaged into chromatin. Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair. Here, we review the methods that have been developed over the years to delineate chromatin alterations in response to DNA damage by focusing on the well-characterized Nucleotide Excision Repair (NER) pathway. We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity. In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair.

The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis.

The accurate repair of DNA double-strand breaks (DSBs), highly toxic DNA lesions, is crucial for genome integrity and is tightly regulated during the cell cycle. In mitosis, cells inactivate DSB repair in favor of a tethering mechanism that stabilizes broken chromosomes until they are repaired in the subsequent cell cycle phases. How this is achieved mechanistically is not yet understood, but the adaptor protein TOPBP1 is critically implicated in this process. Here, we identify CIP2A as a TOPBP1-interacting protein that regulates TOPBP1 localization specifically in mitosis. Cells lacking CIP2A display increased radio-sensitivity, micronuclei formation and chromosomal instability. CIP2A is actively exported from the cell nucleus in interphase but, upon nuclear envelope breakdown at the onset of mitosis, gains access to chromatin where it forms a complex with MDC1 and TOPBP1 to promote TOPBP1 recruitment to sites of mitotic DSBs. Collectively, our data uncover CIP2A-TOPBP1 as a mitosis-specific genome maintenance complex.

The CIP2A-TOPBP1 axis safeguards chromosome stability and is a synthetic lethal target for BRCA-mutated cancer.

BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR-Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers.

Health-related quality of life in long-term prostate cancer survivors after nerve-sparing and non-nerve-sparing radical prostatectomy-Results from the multiregional PROCAS study.

BACKGROUND: Nerve-sparing (NS) surgery was developed to improve postoperative sexual and potentially urological outcomes after radical prostatectomy (RP). However, it is largely unknown how NSRP affects health-related quality of life (HRQoL) including urinary and sexual outcomes in prostate cancer (PC) survivors 5-10 years after diagnosis in comparison with Non-NSRP. METHODS: The study population included 382 stage pT2-T3N0M0 PC survivors 5-10 years post diagnosis, who were identified from the multiregional Prostate Cancer Survivorship in Switzerland (PROCAS) study. Briefly, in 2017/2018, PC survivors were identified via six population-based cancer registries based in both German- and French-speaking Switzerland. HRQoL and PC-specific symptom burden was assessed using the EORTC QLQ-C30 and EORTC QLQ-PR25 questionnaires. Differences in HRQoL outcomes between survivors treated with NSRP (uni- & bilateral) and Non-NSRP were analyzed with multivariable linear regression adjusted for age, years since diagnosis, cancer stage, comorbidities at diagnosis, and further therapies, if appropriate. Multiple imputation was performed to minimize the bias due to missing data. RESULTS: Five to ten years after diagnosis, PC survivors treated with NSRP and Non-NSRP reported similar symptom burden and comparable HRQoL function scores. The only significant differences were reported for sexual activity, whereas PC survivors who underwent NSRP reported statistically significant (P = .031) higher sexual activity than those on Non-NSRP. NSRP and Non-NSRP reported similar scores for urinary symptoms and all other HRQoL outcomes. CONCLUSIONS: Our results support nerve-sparing techniques as an option to improve postoperative sexual, but not urinary outcomes after RP in long-term PC survivors.