Impact of Manganese and Chromate on Specific DNA Double-Strand Break Repair Pathways

Manganese is an essential trace element; nevertheless, on conditions of overload, it becomes toxic, with neurotoxicity being the main concern. Chromate is a well-known human carcinogen. The underlying mechanisms seem to be oxidative stress as well as direct DNA damage in the case of chromate, but also interactions with DNA repair systems in both cases. However, the impact of manganese and chromate on DNA double-strand break (DSB) repair pathways is largely unknown. In the present study, we examined the induction of DSB as well as the effect on specific DNA DSB repair mechanisms, namely homologous recombination (HR), non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ). We applied DSB repair pathway-specific reporter cell lines, pulsed field gel electrophoresis as well as gene expression analysis, and investigated the binding of specific DNA repair proteins via immunoflourescence. While manganese did not seem to induce DNA DSB and had no impact on NHEJ and MMEJ, HR and SSA were inhibited. In the case of chromate, the induction of DSB was further supported. Regarding DSB repair, no inhibition was seen in the case of NHEJ and SSA, but HR was diminished and MMEJ was activated in a pronounced manner. The results indicate a specific inhibition of error-free HR by manganese and chromate, with a shift towards error-prone DSB repair mechanisms in both cases. These observations suggest the induction of genomic instability and may explain the microsatellite instability involved in chromate-induced carcinogenicity

Breaks invisible to the DNA damage response machinery accumulate in ATM-deficient cells

Altres ajuts: Fundació La Marató, Grant number TV32005-050110After irradiation, ATM defective cells accumulate unrepaired double strand breaks (DSBs) for several cell divisions. At the chromosome level, unresolved DSBs appear as chromosome breaks that can be efficiently scored by using telomeric and mFISH probes. H2AX is immediately activated by ATM in response to DNA damage and its phosphorylated form, γH2AX, flanks the DSB through several megabases. The γH2AX-labeling status of broken chromosome ends was analyzed in AT cells to check whether the DNA damage response was accurately taking place in these persistent DSBs. The results show that one quarter of the scored breaks are devoid of γH2AX foci in metaphase spreads from ATM-deficient cells, and this fraction is significantly higher than in normal cells (χ2 < 0.05). Accumulation of sensor and repair proteins at damaged sites is a key event in the cellular response to DSBs, so MRE11 labeling at broken ends was also analyzed. While all γH2AX foci scored at visible broken ends colocalize with MRE11 foci, all γH2AX-unlabeled breaks are also devoid of MRE11-labeling. The present results suggest that a significant subset of the AT long-lived DSBs may persist as " invisible" DSBs due to deficient detection by the DNA damage repair machinery. Eventually the properly signaled DSBs will be repaired while invisible breaks may indefinitely accumulate; most probably contributing to the AT cells' well known genomic instability

Human Research Program Space Radiation Standing Review Panel (SRP)

The Space Radiation Standing Review Panel (SRP) met at the NASA Johnson Space Center (JSC) on December 9-11, 2009 to discuss the areas of current and future research targeted by the Space Radiation Program Element (SRPE) of the Human Research Program (HRP). Using evidence-based knowledge as a background for identified risks to astronaut health and performance, NASA had identified gaps in knowledge to address those risks. Ongoing and proposed tasks were presented to address the gaps. The charge to the Space Radiation SRP was to review the gaps, evaluate whether the tasks addressed these gaps and to make recommendations to NASA s HRP Science Management Office regarding the SRP's review. The SRP was requested to evaluate the practicality of the proposed efforts in light of the demands placed on the HRP. Several presentations were made to the SRP during the site visit and the SRP spent sufficient time to address the SRP charge. The SRP made a final debriefing to the HRP Program Scientist, Dr. John B. Charles, on December 11, 2009. The SRP noted that current SRPE strategy is properly science-based and views this as the best assurance of the likelihood that answers to the questions posed as gaps in knowledge can be found, that the uncertainty in risk estimates can be reduced, and that a solid, cost-effective approach to risk reduction solutions is being developed. The current approach of the SRPE, based on the use of carefully focused research solicitations, requiring thorough peer-review and approaches demonstrated to be on the path to answering the NASA strategic questions, addressed to a broad extramural community of qualified scientists, optimally positioned to take advantage of serendipitous discoveries and to leverage scientific advances made elsewhere, is sound and appropriate. The SRP viewed with concern statements by HRP implying that the only science legitimately deserving support should be "applied" or, in some instances that the very term "research" might be frowned upon. We understand the desire of management to ensure that research stay focused on mission objectives, but the terms used are code words fraught with different meaning for scientists. Such expressions, taken at face value, convey a profoundly flawed view of science, can easily lead down counterproductive paths, and have the potential to irretrievably corrupt NASA requirements. The SRP understands and endorses the mandate to keep research efforts focused on the mission needs. However, thoughtful application of knowledge gained by understanding the mechanisms and pathways of biological effects cannot be replaced

Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

In cells exposed to ionizing radiation (IR), double-strand breaks (DSBs) form within clustered-damage sites from lesions disrupting the DNA sugar–phosphate backbone. It is commonly assumed that these DSBs form promptly and are immediately detected and processed by the cellular DNA damage response (DDR) apparatus. This assumption is questioned by the observation that after irradiation of naked DNA, a fraction of DSBs forms minutes to hours after exposure as a result of temperature dependent, chemical processing of labile sugar lesions. Excess DSBs also form when IR-exposed cells are processed at 50°C, but have been hitherto considered method-related artifact. Thus, it remains unknown whether DSBs actually develop in cells after IR exposure from chemically labile damage. Here, we show that irradiation of ‘naked’ or chromatin-organized mammalian DNA produces lesions, which evolve to DSBs and add to those promptly induced, after 8–24 h in vitro incubation at 37°C or 50°C. The conversion is more efficient in chromatin-associated DNA, completed within 1 h in cells and delayed in a reducing environment. We conclude that IR generates sugar lesions within clustered-damage sites contributing to DSB formation only after chemical processing, which occurs efficiently at 37°C. This subset of delayed DSBs may challenge DDR, may affect the perceived repair kinetics and requires further characterization

No increase in radiation-induced chromosome aberration complexity detected by m-FISH after culture in the presence of 5’-bromodeoxyuridine

The thymidine analogue, 5’-bromodeoxyuridine (BrdU), is a known mutagen that is routinely introduced into culture media for subsequent Harlequin stain analysis and determination of cell cycle status. Previously, we examined the induction of chromosome aberrations in human peripheral blood lymphocytes (PBL) known to be in their 1st cell division following exposure to a low dose (0.5 Gy, average one -particle per cell) of high-LET α-particles. We found complex chromosome aberrations to be characteristic of exposure to high-LET radiation and suggested the features of complex exchange to reflect qualitatively the spatial deposition of this densely ionising radiation. To exclude the possibility that BrdU addition post-irradiation influenced the complexity of chromosomal damage observed by m-FISH, the effect of increasing BrdU concentration on aberration complexity was investigated. Comparisons between BrdU concentration (0, 10, and 40 M) and between sham- and α-particle irradiated PBL, were made both independently and in combination to enable discrimination between BrdU and high-LET radiation effects. Aberration type, size, complexity and completeness were assessed by m-FISH, and the relative progression through cell division was evaluated. We found no evidence of any qualitative difference in the complexity of damage as visualized by m-FISH but did observe an increase in the frequency of complex exchanges with increasing BrdU concentration indicative of altered cell cycle kinetics. The parameters measured here are consistent with findings from previous in vitro and in vivo work, indicating that each complex aberration visualised by m-FISH is characteristic of the structure of the high-LET α-particle track and the geometry of cell irradiated

When a duck is not a duck; a new interdisciplinary synthesis for environmental radiation protection

This consensus paper presents the results of a workshop held in Essen, Germany in September 2017, called to examine critically the current approach to radiological environmental protection. The meeting brought together participants from the field of low dose radiobiology and those working in radioecology. Both groups have a common aim of identifying radiation exposures and protecting populations and individuals from harmful effects of ionising radiation exposure, but rarely work closely together. A key question in radiobiology is to understand mechanisms triggered by low doses or dose rates, leading to adverse outcomes of individuals while in radioecology a key objective is to recognise when harm is occurring at the level of the ecosystem. The discussion provided a total of six strategic recommendations which would help to address these questions.Funding was provided for this workshop by the International Union for Radioecology and the University of Duisburg-Essen

PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer