Ribosome Collisions Trigger General Stress Responses to Regulate Cell Fate.

Problems arising during translation of mRNAs lead to ribosome stalling and collisions that trigger a series of quality control events. However, the global cellular response to ribosome collisions has not been explored. Here, we uncover a function for ribosome collisions in signal transduction. Using translation elongation inhibitors and general cellular stress conditions, including amino acid starvation and UV irradiation, we show that ribosome collisions activate the stress-activated protein kinase (SAPK) and GCN2-mediated stress response pathways. We show that the MAPKKK ZAK functions as the sentinel for ribosome collisions and is required for immediate early activation of both SAPK (p38/JNK) and GCN2 signaling pathways. Selective ribosome profiling and biochemistry demonstrate that although ZAK generally associates with elongating ribosomes on polysomal mRNAs, it specifically auto-phosphorylates on the minimal unit of colliding ribosomes, the disome. Together, these results provide molecular insights into how perturbation of translational homeostasis regulates cell fate.

Deciphering the DNA Damage Response.

This year's Albert Lasker Basic Medical Research Award honors Evelyn Witkin and Stephen J. Elledge, two pioneers in elucidating the DNA damage response, whose contributions span more than 40 years.

Strand-resolved mutagenicity of DNA damage and repair.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.

Inheritance of paternal DNA damage by histone-mediated repair restriction.

How paternal exposure to ionizing radiation affects genetic inheritance and disease risk in the offspring has been a long-standing question in radiation biology. In humans, nearly 80% of transmitted mutations arise in the paternal germline1, but the transgenerational effects of ionizing radiation exposure has remained controversial and the mechanisms are unknown. Here we show that in sex-separated Caenorhabditis elegans strains, paternal, but not maternal, exposure to ionizing radiation leads to transgenerational embryonic lethality. The offspring of irradiated males displayed various genome instability phenotypes, including DNA fragmentation, chromosomal rearrangement and aneuploidy. Paternal DNA double strand breaks were repaired by maternally provided error-prone polymerase theta-mediated end joining. Mechanistically, we show that depletion of an orthologue of human histone H1.0, HIS-24, or the heterochromatin protein HPL-1, could significantly reverse the transgenerational embryonic lethality. Removal of HIS-24 or HPL-1 reduced histone 3 lysine 9 dimethylation and enabled error-free homologous recombination repair in the germline of the F1 generation from ionizing radiation-treated P0 males, consequently improving the viability of the F2 generation. This work establishes the mechanistic underpinnings of the heritable consequences of paternal radiation exposure on the health of offspring, which may lead to congenital disorders and cancer in humans.

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.

Diverse mutational landscapes in human lymphocytes.

The lymphocyte genome is prone to many threats, including programmed mutation during differentiation1, antigen-driven proliferation and residency in diverse microenvironments. Here, after developing protocols for expansion of single-cell lymphocyte cultures, we sequenced whole genomes from 717 normal naive and memory B and T cells and haematopoietic stem cells. All lymphocyte subsets carried more point mutations and structural variants than haematopoietic stem cells, with higher burdens in memory cells than in naive cells, and with T cells accumulating mutations at a higher rate throughout life. Off-target effects of immunological diversification accounted for approximately half of the additional differentiation-associated mutations in lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every on-target IGHV mutation during the germinal centre reaction. Structural variation was 16-fold higher in lymphocytes than in stem cells, with around 15% of deletions being attributable to off-target recombinase-activating gene activity. DNA damage from ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory cells. The mutation burden and signatures of normal B cells were broadly similar to those seen in many B-cell cancers, suggesting that malignant transformation of lymphocytes arises from the same mutational processes that are active across normal ontogeny. The mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.

Crosstalk between Lys63- and Lys11-polyubiquitin signaling at DNA damage sites is driven by Cezanne.

The establishment of polyubiquitin conjugates with distinct linkages play important roles in the DNA damage response. Much remains unknown about the regulation of linkage-specific ubiquitin signaling at sites of DNA damage. Here we reveal that Cezanne (also known as Otud7B) deubiquitinating enzyme promotes the recruitment of Rap80/BRCA1-A complex by binding to Lys63-polyubiquitin and targeting Lys11-polyubiquitin. Using a ubiquitin binding domain protein array screen, we identify that the UBA domains of Cezanne and Cezanne2 (also known as Otud7A) selectively bind to Lys63-linked polyubiquitin. Increased Lys11-linkage ubiquitination due to lack of Cezanne DUB activity compromises the recruitment of Rap80/BRCA1-A. Cezanne2 interacts with Cezanne, facilitating Cezanne in the recruitment of Rap80/BRCA1-A, Rad18, and 53BP1, in cellular resistance to ionizing radiation and DNA repair. Our work presents a model that Cezanne serves as a "reader" of the Lys63-linkage polyubiquitin at DNA damage sites and an "eraser" of the Lys11-linkage ubiquitination, indicating a crosstalk between linkage-specific ubiquitination at DNA damage sites.

Unraveling the Complexity of DNA Radiation Damage Using DNA Nanotechnology.

Radiation cancer therapies use different ionizing radiation qualities that damage DNA molecules in tumor cells by a yet not completely understood plethora of mechanisms and processes. While the direct action of the radiation is significant, the byproducts of the water radiolysis, mainly secondary low-energy electrons (LEEs, <20 eV) and reactive oxygen species (ROS), can also efficiently cause DNA damage, in terms of DNA strand breakage or DNA interstrand cross-linking. As a result, these types of DNA damage evolve into mutations hindering DNA replication, leading to cancer cell death. Concomitant chemo-radiotherapy explores the addition of radiosensitizing therapeutics commonly targeting DNA, such as platinum derivatives and halogenated nucleosides, to enhance the harmful effects of ionizing radiation on the DNA molecule. Further complicating the landscape of DNA damage are secondary structures such as G-quadruplexes occurring in telomeric DNA. These structures protect DNA from radiation damage, rendering them as promising targets for new and more selective cancer radiation treatments, rather than targeting linear DNA. However, despite extensive research, there is no single paradigm approach to understanding the mysterious way in which ionizing radiation causes DNA damage. This is due to the multidisciplinary nature of the field of research, which deals with multiple levels of biological organization, from the molecular building blocks of life toward cells and organisms, as well as with complex multiscale radiation-induced effects. Also, intrinsic DNA features, such as DNA topology and specific oligonucleotide sequences, strongly influence its response to damage from ionizing radiation. In this Account, we present our studies focused on the absolute quantification of photon- and low-energy electron-induced DNA damage in strategically selected target DNA sequences. Our methodology involves using DNA origami nanostructures, specifically the Rothemund triangle, as a platform to expose DNA sequences to either low-energy electrons or vacuum-ultraviolet (VUV, <15 eV) photons and subsequent atomic force microscopy (AFM) analysis. Through this approach, the effects of the DNA sequence, incorporation of halogenated radiosensitizers, DNA topology, and the radiation quality on radiation-induced DNA strand breakage have been systematically assessed and correlated with fundamental photon- and electron-driven mechanisms underlying DNA radiation damage. At lower energies, these mechanisms include dissociative electron attachment (DEA), where electrons attach to DNA molecules causing strand breaks, and dissociative photoexcitation of DNA. Additionally, further dissociative processes such as photoionization and electron impact contribute to the complex cascade of DNA damage events induced by ionizing radiation. We expect that emerging DNA origami-based approaches will lead to a paradigm shift in research fields associated with DNA damage and suggest future directions, which can foster the development of technological applications in nanomedicine, e.g., optimized cancer treatments or the molecular design of optimized radiosensitizing therapeutics.

The UVSSA protein is part of a genome integrity homeostasis network with links to transcription-coupled DNA repair and ATM signaling.

SignificanceTranscription-coupled repair (TCR) involves four core proteins: CSA, CSB, USP7, and UVSSA. CSA and CSB are mutated in the severe human neurocutaneous disease Cockayne syndrome. In contrast UVSSA is a mild photosensitive disease in which a mutated protein sequence prevents recruitment of USP7 protease to deubiquitinate and stabilize CSB. We deleted the UVSSA protein using CRISPR-Cas9 in an aneuploid cell line, HEK293, and determined the functional consequences. The knockout cell line was sensitive to transcription-blocking lesions but not sensitive to oxidative agents or PARP inhibitors, unlike CSB. Knockout of UVSSA also activated ATM, like CSB, in transcription-arrested cells. The phenotype of UVSSA, especially its rarity, suggests that many TCR-deficient patients and tumors fail to be recognized clinically.

A genome-wide screen reveals that Dyrk1A kinase promotes nucleotide excision repair by preventing aberrant overexpression of cyclin D1 and p21.

Nucleotide excision repair (NER) eliminates highly genotoxic solar UV-induced DNA photoproducts that otherwise stimulate malignant melanoma development. Here, a genome-wide loss-of-function screen, coupling CRISPR/Cas9 technology with a flow cytometry-based DNA repair assay, was used to identify novel genes required for efficient NER in primary human fibroblasts. Interestingly, the screen revealed multiple genes encoding proteins, with no previously known involvement in UV damage repair, that significantly modulate NER uniquely during S phase of the cell cycle. Among these, we further characterized Dyrk1A, a dual specificity kinase that phosphorylates the proto-oncoprotein cyclin D1 on threonine 286 (T286), thereby stimulating its timely cytoplasmic relocalization and proteasomal degradation, which is required for proper regulation of the G1-S phase transition and control of cellular proliferation. We demonstrate that in UV-irradiated HeLa cells, depletion of Dyrk1A leading to overexpression of cyclin D1 causes inhibition of NER uniquely during S phase and reduced cell survival. Consistently, expression/nuclear accumulation of nonphosphorylatable cyclin D1 (T286A) in melanoma cells strongly interferes with S phase NER and enhances cytotoxicity post-UV. Moreover, the negative impact of cyclin D1 (T286A) overexpression on repair is independent of cyclin-dependent kinase activity but requires cyclin D1-dependent upregulation of p21 expression. Our data indicate that inhibition of NER during S phase might represent a previously unappreciated noncanonical mechanism by which oncogenic cyclin D1 fosters melanomagenesis.

Natural Epigenetic DNA Modifications Cause Remote DNA Photodamage.

5-Formyl-2'-deoxycytidine, an intermediate during the erasure of epigenetic marker 5-methyl-2'-deoxycytidine, and 5-formyl-2'-deoxyuridine, an oxidative lesion of thymidine, are naturally occurring DNA modifications. The carbonyl groups of these DNA modifications are the smallest possible photosensitizers and have the potential to generate cyclobutane pyrimidine dimers upon irradiation with UV light. To evidence this damaging potential, ternary DNA architectures were used, in which the photosensitizer and the damage site were located at well-defined positions in the sequences. The quantitative and time-dependent analysis revealed not only the high photodamaging potential of both natural DNA modifications but also the mechanisms for this new pathway to photodamage. 5-Formyl-2'-deoxycytidine is more efficiently generating cyclobutane pyrimidine dimers than 5-formyl-2'-deoxyuridine because the latter is also photochemically converted to 5-carboxy-2'-deoxyuridine. This demonstrates for the first time that epigenetic DNA modifications regulating gene expression interact with sunlight and can induce DNA photodamages.

Upregulation of DNA repair genes and cell extrusion underpin the remarkable radiation resistance of Trichoplax adhaerens.

Trichoplax adhaerens is the simplest multicellular animal with tissue differentiation and somatic cell turnover. Like all other multicellular organisms, it should be vulnerable to cancer, yet there have been no reports of cancer in T. adhaerens or any other placozoan. We investigated the cancer resistance of T. adhaerens, discovering that they are able to tolerate high levels of radiation damage (218.6 Gy). To investigate how T. adhaerens survive levels of radiation that are lethal to other animals, we examined gene expression after the X-ray exposure, finding overexpression of genes involved in DNA repair and apoptosis including the MDM2 gene. We also discovered that T. adhaerens extrudes clusters of inviable cells after X-ray exposure. T. adhaerens is a valuable model organism for studying the molecular, genetic, and tissue-level mechanisms underlying cancer suppression.

Impact of double-strand breaks induced by uv radiation on neuroinflammation and neurodegenerative disorders.

Exposure to UV affects the development and growth of a wide range of organisms. Nowadays, researchers are focusing on the impact of UV radiation and its underlying molecular mechanisms, as well as devising strategies to mitigate its harmful effects. Different forms of UV radiation, their typical exposure effects, the impact of UV on DNA integrity, and the deterioration of genetic material are discussed in this review; furthermore, we also review the effects of UV radiation that affect the biological functions of the organisms. Subsequently, we address the processes that aid organisms in navigating the damage in genetic material, neuroinflammation, and neurodegeneration brought on by UV-mediated double-strand breaks. To emphasize the molecular pathways, we conclude the review by going over the animal model studies that highlight the genes and proteins that are impacted by UV radiation.

Protection from UV light is an evolutionarily conserved feature of the haematopoietic niche.

Haematopoietic stem and progenitor cells (HSPCs) require a specific microenvironment, the haematopoietic niche, which regulates HSPC behaviour1,2. The location of this niche varies across species, but the evolutionary pressures that drive HSPCs to different microenvironments remain unknown. The niche is located in the bone marrow in adult mammals, whereas it is found in other locations in non-mammalian vertebrates, for example, in the kidney marrow in teleost fish. Here we show that a melanocyte umbrella above the kidney marrow protects HSPCs against ultraviolet light in zebrafish. Because mutants that lack melanocytes have normal steady-state haematopoiesis under standard laboratory conditions, we hypothesized that melanocytes above the stem cell niche protect HSPCs against ultraviolet-light-induced DNA damage. Indeed, after ultraviolet-light irradiation, unpigmented larvae show higher levels of DNA damage in HSPCs, as indicated by staining of cyclobutane pyrimidine dimers and have reduced numbers of HSPCs, as shown by cmyb (also known as myb) expression. The umbrella of melanocytes associated with the haematopoietic niche is highly evolutionarily conserved in aquatic animals, including the sea lamprey, a basal vertebrate. During the transition from an aquatic to a terrestrial environment, HSPCs relocated into the bone marrow, which is protected from ultraviolet light by the cortical bone around the marrow. Our studies reveal that melanocytes above the haematopoietic niche protect HSPCs from ultraviolet-light-induced DNA damage in aquatic vertebrates and suggest that during the transition to terrestrial life, ultraviolet light was an evolutionary pressure affecting the location of the haematopoietic niche.

Efficacy of Combinational Treatment versus Nicotinamide Monotherapy in the Prevention of Ultraviolet Radiation-Induced Skin Cancer.

INTRODUCTION: Ultraviolet radiation (UVR) is the primary risk factor for keratinocyte carcinomas. Oral supplementation with nicotinamide (NAM) is reported to reduce the formation of new keratinocyte carcinomas. NAM's photoprotection is mediated by enhanced DNA repair. We wanted to explore whether NAM in combination with antiproliferative (metformin [Met]) or antioxidant (phloroglucinol [PG]) compounds could potentially enhance its photoprotective effects. METHODS: Hairless mice (C3.Cg-Hrhr/TifBomTac) were treated orally with either a standard dose of NAM monotherapy (NAM-mono; 600 mg/kg) or NAM (400 mg/kg) combined with Met (200 mg/kg) (NAM-Met) or PG (75 mg/kg) (NAM-PG). Mice were irradiated with 3.5 standard erythema doses of UVR three times per week to induce tumour development. Photoprotective effects were based on (i) tumour onset of the first three tumours, (ii) skin photodamage, and (iii) DNA damage (cyclobutane pyrimidine dimers [CPDs] and pyrimidine-pyrimidone (6-4) photoproducts [6-4PPs]). RESULTS: All mice treated with NAM demonstrated a delay in tumour onset and reduced tumour burden compared to the UV control group (NAM, NAM-Met, NAM-PG vs. UV control: p ≤ 0.015). NAM-mono and NAM-PG increased time until all three tumours with no difference between them, indicating a similar degree of photoprotection. NAM-mono had no effect on DNA damage compared to the UV control group (p > 0.05), whereas NAM-PG reduced 6-4PP lesions (p < 0.01) but not CPDs (p > 0.05) compared to NAM-mono. NAM-Met delayed the onset of the third tumour compared to the UV control but demonstrated a quicker onset compared to NAM-mono, suggesting inferior photoprotection compared to nicotinamide monotherapy. CONCLUSION: NAM-PG was as effective in delaying UVR-induced tumour onset as NAM-mono. The reduction in 6-4PP lesions may indicate that the mechanism of NAM-PG is better suited for photoprotection than NAM-mono. NAM-mono was superior to NAM-Met, indicating a dose dependency of NAM's photoprotection. These results highlight the potential for combining photoprotective compounds to enhance photoprotection.

Thymine dissociation and dimer formation: A Raman and synchronous fluorescence spectroscopic study.

In this study, absorption, fluorescence, synchronous fluorescence, and Raman spectra of nonirradiated and ultraviolet (UV)-irradiated thymine solutions were recorded in order to detect thymine dimer formation. The thymine dimer formation, as a function of irradiation dose, was determined by Raman spectroscopy. In addition, the formation of a mutagenic (6-4) photoproduct was identified by its synchronous fluorescence spectrum. Our spectroscopic data suggest that the rate of conversion of thymine to thymine dimer decreases after 20 min of UV irradiation, owing to the formation of an equilibrium between the thymine dimers and monomers. However, the formation of the (6-4) photoproduct continued to increase with UV irradiation. In addition, the Raman spectra of nonirradiated and irradiated calf thymus DNA were recorded, and the formation of thymine dimers was detected. The spectroscopic data presented make it possible to determine the mechanism of thymine dimer formation, which is known to be responsible for the inhibition of DNA replication that causes bacteria inactivation.

UV-exposure, endogenous DNA damage, and DNA replication errors shape the spectra of genome changes in human skin.

Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.

Mdm2 phosphorylation by Akt regulates the p53 response to oxidative stress to promote cell proliferation and tumorigenesis.

We have shown previously that phosphorylation of Mdm2 by ATM and c-Abl regulates Mdm2-p53 signaling and alters the effects of DNA damage in mice, including bone marrow failure and tumorigenesis induced by ionizing radiation. Here, we examine the physiological effects of Mdm2 phosphorylation by Akt, another DNA damage effector kinase. Surprisingly, Akt phosphorylation of Mdm2 does not alter the p53-mediated effects of ionizing radiation in cells or mice but regulates the p53 response to oxidative stress. Akt phosphorylation of Mdm2 serine residue 183 increases nuclear Mdm2 stability, decreases p53 levels, and prevents senescence in primary cells exposed to reactive oxidative species (ROS). Using multiple mouse models of ROS-induced cancer, we show that Mdm2 phosphorylation by Akt reduces senescence to promote KrasG12D-driven lung cancers and carcinogen-induced papilloma and hepatocellular carcinomas. Collectively, we document a unique physiologic role for Akt-Mdm2-p53 signaling in regulating cell growth and tumorigenesis in response to oxidative stress.

Occupational Risks of Radiation Exposure to Cardiologists.

PURPOSE OF REVIEW: Invasive cardiologists are exposed to large amounts of ionizing radiation. This review aims to summarize the main occupational risks in a radiation-exposed cardiology practice. RECENT FINDINGS: We carried out a literature review on the subject. The studies reviewed allowed us to list six main health risk categories possibly associated with radiation exposure among cardiologists: deoxyribonucleic acid (DNA) and biochemical damages; cancers; ocular manifestations; olfaction, vascular, and neuropsychological alterations; musculoskeletal problems; and reproductive risks. Our descriptive analysis demonstrates higher risks of DNA damage and lens opacities among radiation-exposed cardiology staff. Surveys and questionnaires have demonstrated a higher risk of musculoskeletal disease in exposed workers. Studies reported no difference in cancer frequency between radiation-exposed workers and controls. Changes in olfactory performance, neuropsychological aspects, and vascular changes have also been reported. Limited literature supports the security of continuing radiation-exposed work during pregnancy. Therefore, there is an urgent need to increase knowledge of the occupational risks of radiation exposure and to adopt technologies to reduce them.

DNA-damage RBE assessment for combined boron and gadolinium neutron capture therapy.

PURPOSE: Neutron capture therapy (NCT) by 10B and 157Gd agents is a unique irradiation-based method which can be used to treat brain tumors. Current study aims to quantitatively evaluate the relative biological effectiveness (RBE) and dose distributions during the combined BNCT and GdNCT modalities through a hybrid Monte Carlo (MC) simulation approach. METHODS: Snyder head phantom as well as a cubic hypothetical tumor was at first modeled by Geant4 MC Code. Then, the energy spectra and dose distribution relevant to the released secondary particles during the combined Gd/BNCT were scored for different concentrations of 157Gd and 10B inside tumor volume. Finally, the scored energy spectra were imported to the MCDS code to estimate both RBESSB and RBEDSB values for different 157Gd concentrations. RESULTS: The results showed that combined Gd/BNCT increases the fluence-averaged RBESSB values by about 1.7 times when 157Gd concentration increments from 0 to 2000 µg/g for both considered cell oxygen levels (pO2 = 10% and 100%). Besides, a reduction of about 26% was found for fluence-averaged RBEDSB values with an increment of 157Gd concentration in tumor volume. CONCLUSION: From the results, it can be concluded that combined Gd/BNCT technique can improve tumor coverage with higher dose levels but in the expense of RBEDSB reduction which can affect the clinical efficacy of the NCT technique.