Results for “DNA Repair” — Page 11

NASA NTRS2001

Clustered DNA damages induced by high and low LET radiation, including heavy ions

Clustered DNA damages--here defined as two or more lesions (strand breaks, oxidized purines, oxidized pyrimidines or abasic sites) within a few helical turns--have been postulated as difficult to repair accurately, and thus highly significant biological lesions. Further, attempted repair of clusters may produce double strand breaks (DSBs). However, until recently, there was no way to measure ionizing radiation-induced clustered damages, except DSB. We recently described an approach for measuring classes of clustered damages (oxidized purine clusters, oxidized pyrimidine clusters, abasic clusters, along with DSB). We showed that ionizing radiation (gamma rays and Fe ions, 1 GeV/amu) does induce such clusters in genomic DNA in solution and in human cells. These studies also showed that each damage cluster results from one radiation hit (and its track), thus indicating that they can be induced by very low doses of radiation, i.e. two independent hits are not required for cluster induction. Further, among all complex damages, double strand breaks comprise--at most-- ~20%, with the other clustered damages being at least 80%.

NASA Discipline Radiation Health↗

NASA NTRS2007

Stochastic Effects in Computational Biology of Space Radiation Cancer Risk

Estimating risk from space radiation poses important questions on the radiobiology of protons and heavy ions. We are considering systems biology models to study radiation induced repair foci (RIRF) at low doses, in which less than one-track on average transverses the cell, and the subsequent DNA damage processing and signal transduction events. Computational approaches for describing protein regulatory networks coupled to DNA and oxidative damage sites include systems of differential equations, stochastic equations, and Monte-Carlo simulations. We review recent developments in the mathematical description of protein regulatory networks and possible approaches to radiation effects simulation. These include robustness, which states that regulatory networks maintain their functions against external and internal perturbations due to compensating properties of redundancy and molecular feedback controls, and modularity, which leads to general theorems for considering molecules that interact through a regulatory mechanism without exchange of matter leading to a block diagonal reduction of the connecting pathways. Identifying rate-limiting steps, robustness, and modularity in pathways perturbed by radiation damage are shown to be valid techniques for reducing large molecular systems to realistic computer simulations. Other techniques studied are the use of steady-state analysis, and the introduction of composite molecules or rate-constants to represent small collections of reactants. Applications of these techniques to describe spatial and temporal distributions of RIRF and cell populations following low dose irradiation are described.

Cucinotta, Francis A.↗

NASA NTRS2000

Radiation-induced heat-labile sites that convert into DNA double-strand breaks

The yield of DNA double-strand breaks (DSBs) in SV40 DNA irradiated in aqueous solution was found to increase by more than a factor of two as a result of postirradiation incubation of the DNA at 50 degrees C and pH 8.0 for 24 h. This is in agreement with data from studies performed at 37 degrees C that were published previously. Importantly, similar results were also obtained from irradiation of mammalian DNA in agarose plugs. These results suggest that heat-labile sites within locally multiply damaged sites are produced by radiation and are subsequently transformed into DSBs. Since incubation at 50 degrees C is typically employed for lysis of cells in commonly used pulsed-field gel assays for detection of DSBs in mammalian cells, the possibility that heat-labile sites are present in irradiated cells was also studied. An increase in the apparent number of DSBs as a function of lysis time at 50 degrees C was found with kinetics that was similar to that for irradiated DNA, although the magnitude of the increase was smaller. This suggests that heat-labile sites are also formed in the cell. If this is the case, a proportion of DSBs measured by the pulsed-field gel assays may occur during the lysis step and may not be present in the cell as breaks but as heat-labile sites. It is suggested that such sites consist mainly of heat-labile sugar lesions within locally multiply damaged sites. Comparing rejoining of DSBs measured with short and long lysis procedure indicates that the heat-labile sites are repaired with fast kinetics in comparison with repair of the bulk of DSBs.

Non-NASA Center↗

NASA NTRS2007

Chromosome Aberrations in Normal and Ataxia-Telangiectasia Cells Exposed to Heavy Ions

Although cells derived from Ataxia Telangiectasia (AT) patients are known to exhibit abnormal responses to ionizing radiations, its underlying mechanism still remains unclear. Previously, the authors reported that at the same gamma-irradiation dose AT cells show higher frequencies of misrepair and deletions compared to normal human fibroblast cells. In this study, we investigated the effects of heavy ions beams on chromosomal aberrations in normal and AT cells. Normal and AT fibroblast cells arrested at G0/G1 phase were irradiated with 2 Gy of X-rays, 490 MeV/u Silicon (LET 55 keV/m), 500 MeV/u Iron (LET 185 keV/m) and 200 MeV/u Iron (LET 440 keV/m) particles, and then cells were allowed to repair for 24 hours at 37 degrees before subculture. Calyculin-A induced PCC method was employed to collect G2/M chromosomes and whole DNA probes 1 and 3 were used to analyze chromosomal aberrations such as color-junctions, deletions, simple exchanges (incomplete and reciprocal exanges) and complex-type exchanges. The percentages of aberrant cells were higher when normal and AT cells were exposed to heavy ions compared to X-rays, and had a tendency to increase with increasing LET up to 185 keV/m and then decreased at 440 keV/m. When the frequency of color-junctions per cell was compared after X-ray exposure, AT cells had around three times higher frequency of color-junctions (mis-rejoining) than normal cells. However, at 185 keV/m there was no difference in the frequency of color-junctions between two cell lines. It was also found that the frequency of simple exchanges per cell was almost constant in AT cells regardless LET levels, but it was LET dependent for normal cells. Interestingly, the frequency of simple exchanges was higher for AT cells when it was compared at 185 keV/m but AT cells had more complex-type exchanges at the same LET levels. Heavy ions are more efficient in inducing chromosome aberrations in normal and AT cells compared to X-rays, and the aberration types between normal and AT fibroblast appeared different probably due to difference in the ATM gene function.

Kawata, T.↗

NASA NTRS1995

Deletions at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system

Using the radiomimetic drug, bleomycin, we have determined the mutagenic potential of DNA strand breaks in the shuttle vector pZ189 in human fibroblasts. The bleomycin treatment conditions used produce strand breaks with 3'-phosphoglycolate termini as > 95% of the detectable dose-dependent lesions. Breaks with this end group represent 50% of the strand break damage produced by ionizing radiation. We report that such strand breaks are mutagenic lesions. The type of mutation produced is largely determined by the type of strand break on the plasmid (i.e. single versus double). Mutagenesis studies with purified DNA forms showed that nicked plasmids (i.e. those containing single-strand breaks) predominantly produce base substitutions, the majority of which are multiples, which presumably originate from error-prone polymerase activity at strand break sites. In contrast, repair of linear plasmids (i.e. those containing double-strand breaks) mainly results in deletions at short direct repeat sequences, indicating the involvement of illegitimate recombination. The data characterize the nature of mutations produced by single- and double-strand breaks in human cells, and suggests that deletions at direct repeats may be a 'signature' mutation for the processing of DNA double-strand breaks.

NASA Discipline Radiation Health↗

NASA NTRS2002

Clustered DNA damages induced in human hematopoietic cells by low doses of ionizing radiation

Ionizing radiation induces clusters of DNA damages--oxidized bases, abasic sites and strand breaks--on opposing strands within a few helical turns. Such damages have been postulated to be difficult to repair, as are double strand breaks (one type of cluster). We have shown that low doses of low and high linear energy transfer (LET) radiation induce such damage clusters in human cells. In human cells, DSB are about 30% of the total of complex damages, and the levels of DSBs and oxidized pyrimidine clusters are similar. The dose responses for cluster induction in cells can be described by a linear relationship, implying that even low doses of ionizing radiation can produce clustered damages. Studies are in progress to determine whether clusters can be produced by mechanisms other than ionizing radiation, as well as the levels of various cluster types formed by low and high LET radiation.

NASA Discipline Radiation Health↗

NASA NTRS2009

KSC Tech Transfer News, Volume 2, No. 2

This issue contains articles about: (1) the Innovative Partnerships Program (IPP) and the manager of the program, Alexis Hongamen, (2) New Technology Report (NTR) on a Monte Carlo Simulation to Estimate the Likelihood of Direct Lightning Strikes, (3) Kennedy Space Center's Applied Physics Lab, (4) a virtual ruler that is used for many applications, (5) a portable device that finds low-level leaks, (6) a sun-shield, that supports in-space cryogenic propellant storage, (7) lunar dust modeling software, (8) space based monitoring of radiation damage to DNA, (9) the use of light-emitting diode (LED) arrays vegetable production system, (10) Dust Tolerant Intelligent Electrical Connection Systems, (11) Ice Detection Camera System Upgrade, (12) Repair Techniques for Composite Structures, (13) Cryogenic Orbital Testbed, and (14) copyright protection.

Makufka, David↗

NASA NTRS2001

Evidence for factors modulating radiation-induced G2-delay: potential application as radioprotectors

Manipulation of checkpoint response to DNA damage can be developed as a means for protecting astronauts from the adverse effects of unexpected, or background exposures to ionizing radiation. To achieve this goal reagents need to be developed that protect cells from radiation injury by prolonging checkpoint response, thus promoting repair. We present evidence for a low molecular weight substance excreted by cells that dramatically increases the duration of the G2-delay. This compound is termed G2-Arrest Modulating Activity (GAMA). A rat cell line (A1-5) generated by transforming rat embryo fibroblasts with a temperature sensitive form of p53 plus H-ras demonstrates a dramatic increase in radiation resistance after exposure to low LET radiation that is not associated with an increase in the efficiency of rejoining of DNA double strand breaks. Radioresistance in this cell line correlates with a dramatic increase in the duration of the G2 arrest that is modulated by a GAMA produced by actively growing cells. The properties of GAMA suggest that it is a low molecular weight heat-stable peptide. Further characterization of this substance and elucidation of its mechanism of action may allow the development of a biological response modifier with potential applications as a radioprotector. GAMA may be useful for protecting astronauts from radiation injury as preliminary evidence suggests that it is able to modulate the response of cells exposed to heavy ion radiation, similar to that encountered in outer space.

NASA Discipline Radiation Health↗

NASA NTRS1982

Is cell aging caused by respiration-dependent injury to the mitochondrial genome

Though intrinsic mitochondrial aging has been considered before as a possible cause of cellular senescence, the mechanisms of such mitochondrial aging have remained obscure. In this article, the hypothesis of free-radical-induced inhibition of mitochondrial replenishment in fixed postmitotic cells is expanded. It is maintained that the respiration-dependent production of superoxide and hydroxyl radicals may not be fully counteracted, leading to a continuous production of lipoperoxides and malonaldehyde in actively respiring mitochondria. These compounds, in turn, can easily react with the mitochondrial DNA which is in close spatial relationship with the inner mitochondrial membrane, producing an injury that the mitochondria may be unable to counteract because of their apparent lack of adequate repair mechanisms. Mitochondrial division may thus be inhibited leading to age-related reduction of mitochondrial numbers, a deficit in energy production with a concomitant decrease in protein synthesis, deterioration of physiological performance, and, therefore, of organismic performance.

Fleming, J. E.↗

NASA NTRS2018

Computational Model Prediction and Biological Validation Using Simplified Mixed Field Exposures for the Development of a GCR Reference Field

The yield of chromosomal aberrations has been shown to increase in the lymphocytes of astronauts after long-duration missions of several months in space. Chromosome exchanges, especially translocations, are positively correlated with many cancers and are therefore a potential biomarker of cancer risk associated with radiation exposure. Although extensive studies have been carried out on the induction of chromosomal aberrations by low- and high-LET radiation in human lymphocytes, fibroblasts, and epithelial cells exposed in vitro, there is a lack of data on chromosome aberrations induced by low dose-rate chronic exposure and mixed field beams such as those expected in space. Chromosome aberration studies at NSRL will provide the biological validation needed to extend the computational models over a broader range of experimental conditions (more complicated mixed fields leading up to the galactic cosmic rays (GCR) simulator), helping to reduce uncertainties in radiation quality effects and dose-rate dependence in cancer risk models. These models can then be used to answer some of the open questions regarding requirements for a full GCR reference field, including particle type and number, energy, dose rate, and delivery order. In this study, we designed a simplified mixed field beam with a combination of proton, helium, oxygen, and iron ions with shielding or proton, helium, oxygen, and titanium without shielding. Human fibroblasts cells were irradiated with these mixed field beam as well as each single beam with acute and chronic dose rate, and chromosome aberrations (CA) were measured with 3-color fluorescent in situ hybridization (FISH) chromosome painting methods. Frequency and type of CA induced with acute dose rate and chronic dose rates with single and mixed field beam will be discussed. A computational chromosome and radiation-induced DNA damage model, BDSTRACKS (Biological Damage by Stochastic Tracks), was updated to simulate various types of CA induced by acute exposures of the mixed field beams used for the experiments. The chromosomes were simulated by a polymer random walk algorithm with restrictions to their respective domains in the nucleus [1]. The stochastic dose to the nucleus was calculated with the code RITRACKS [2]. Irradiation of a target volume by a mixed field of ions was implemented within RITRACKs, and the fields of ions can be delivered over specific periods of time, allowing the simulation of dose-rate effects. Similarly, particles of various types and energies extracted from a pre-calculated spectra of galactic cosmic rays (GCR) can be used in RITRACKS. The number and spatial location of DSBs (DNA double-strand breaks) were calculated in BDSTRACKS using the simulated chromosomes and local (voxel) dose. Assuming that DSBs led to chromosome breaks, and simulating the rejoining of damaged chromosomes occurring during repair, BDSTRACKS produces the yield of various types of chromosome aberrations as a function of time (only final yields are presented). A comparison between experimental and simulation results will be shown.

Hada, M.↗

NASA NTRS1989

Neoplastic cell transformation by high-LET radiation - Molecular mechanisms

Quantitative data were collected on dose-response curves of cultured mouse-embryo cells (C3H10T1/2) irradiated with heavy ions of various charges and energies. Results suggests that two breaks formed on DNA within 80 A may cause cell transformation and that two DNA breaks formed within 20 A may be lethal. From results of experiments with restriction enzymes which produce DNA damages at specific sites, it was found that DNA double strand breaks are important primary lesions for radiogenic cell transformation and that blunt-ended double-strand breaks can form lethal as well as transformational damages due to misrepair or incomplete repair in the cell. The RBE-LET relationship for high-LET radiation is similar to that for HGPRT locus mutation, chromosomal deletion, and cell transformation, indicating that common lesions may be involved in these radiation effects.

Yang, Tracy Chui-Hsu↗

NASA NTRS1978

The effect of hyperthermia on the radiation response of crypt cells in mouse jejunum

The effect of hyperthermia and/or gamma-radiation on the survival of intestinal crypt cells was studied in BDF sub 1 mice using a microcolony assay. Hyperthermia treatments, which in themselves caused no detectable cell lethality, inhibited the capacity of crypt cells to repair sublethal radiation damage. In addition, heat applied either before or after single radiation exposures potentiated lethal damage to crypt cells; the degree of enhancement was dependent on the time interval between treatments. At the levels of heating employed, DNA synthesis in the intestinal epithelium was significantly reduced immediately following exposure, but returned rapidly to normal levels. No further disturbances in cellular kinetics were observed for up to 10 days after heating.

Wilson, J. D.↗

NASA NTRS2024

ISS External Microorganisms: A Planetary Protection Experiment to Inform Requirements for Crewed Missions to Mars

We have developed, tested, and flown a caddy capable of collecting aseptic samples from external surfaces of the ISS (International Space Station). The sampling caddy is certified for use during US EVA (extra vehicular activity) and was launched to ISS in the summer of 2023. We are scheduled to collect samples from 6 locations outside ISS during an EVA in May of 2024. We will freeze these samples at -80°C on orbit and return them to Earth. We will then extract and sequence any DNA collected during the EVA using next generation sequencing technologies to characterize the community composition and function of each sample. Measuring the type and quantity of microbes present on the exterior of ISS will allow us to address knowledge gap 2B, “Acceptable levels of microbial/organic releases from humans and support systems” described in a 2019 COSPAR report. Collecting data about microbial release from current crewed vehicles will inform requirements for acceptable leak rates for future crewed missions to Mars. The sampling kit consists of eight commercially available, sterile, DNA free, macrofoam swabs ( 23 mm. diameter ) installed in custom aluminum end effectors. Each end effector is housed in and individual aluminum canister. Each canister contains a 0.2 μm Teflon filter to allow the interior volume to accommodate pressure changes without permitting microbial contaminants to enter the sterile interior volume. A handle repurposed from the space shuttle tile repair kit is used to remove the end effector from the sample canister, collect a sample by swabbing a surface and then replace the end effector in its canister. The canisters and end effectors were cleaned and assembled on Earth. Prior to installing the sterile swab the canisters and end effectors were sterilized in an autoclave at 134°C, 215 kPA, for 7 min.. The final assembly occurred in a sterilized class II biosafety cabinet. We will collect six samples from the 1) airlock vestibule, 2) airlock thermal cover, 3) a gap in the micrometeorite shielding near the airlock, 4) a handrail near the airlock, 5) the CDRA (Carbon Dioxide Removal Assembly) vent, and 6) the VES (Vacuum Exhaust System) vent. The remaining two swabs will be reserved as controls. One swab will be exposed during the EVA without touching any surfaces to act as a blank. The final swab will remain sealed until the entire sampling kit is returned to earth. Based on previously published results from the Russian segment, we hypothesize that there will be detectable microbes at some or all of these locations. Ground-based testing of this sampling caddy confirms that the swabs remain sterile as the canisters transition in and out of vacuum. We were able to retrieve, viable bacterial and fungal cells as well as DNA from samples collected from US space suits during vacuum chamber tests lasting as long as seven hours. Based on these results and feedback from the test subjects the sampling caddy was modified to improve ergonomics and meet US EVA safety requirements. Bayonet probes were added to the sides of the sample kit as alternate mounting points. Additional locking features were added to the end effector and the filter stack to prevent inadvertent release during use. The opening mechanism was changed from one where the end effector was rocked laterally to defeat a ball detent to a twist-to-open threaded closure for similar reasons. Demonstrating, this sampling caddy’s effectiveness during a US EVA will allow us to address knowledge gaps identified in COSPAR reports and begin to define planetary protection requirements for life support systems on crewed missions to mars. This kit could also be used to collect contamination control samples during Artemis missions to verify requirements and could be easily modified for robotic sample collection.

Planetary Protection↗

NASA NTRSDate not supplied

ISS External Microorganisms: Collecting Planetary Protection Samples During Extravehicular Activity

We have developed, tested, and flown a caddy capable of collecting aseptic samples from external surfaces of the ISS (International Space Station). The sampling caddy is certified for use during US EVA (extra vehicular activity) and was launched to ISS in the summer of 2023. We are scheduled to collect samples from 6 locations outside ISS during an EVA in May of 2024. We will freeze these samples at -80°C on orbit and return them to Earth. We will then extract and sequence any DNA collected during the EVA using next generation sequencing technologies to characterize the community composition and function of each sample. Measuring the type and quantity of microbes present on the exterior of ISS will allow us to address knowledge gap 2B, “Acceptable levels of microbial/organic releases from humans and support systems” described in a 2019 COSPAR report. Collecting data about microbial release from current crewed vehicles will inform requirements for acceptable leak rates for future crewed missions to Mars. The sampling kit consists of eight commercially available, sterile, DNA free, macrofoam swabs ( 23 mm. diameter ) installed in custom aluminum end effectors. Each end effector is housed in and individual aluminum canister. Each canister contains a 0.2 μm Teflon filter to allow the interior volume to accommodate pressure changes without permitting microbial contaminants to enter the sterile interior volume. A handle repurposed from the space shuttle tile repair kit is used to remove the end effector from the sample canister, collect a sample by swabbing a surface and then replace the end effector in its canister. The canisters and end effectors were cleaned and assembled on Earth. Prior to installing the sterile swab the canisters and end effectors were sterilized in an autoclave at 134°C, 215 kPA, for 7 min.. The final assembly occurred in a sterilized class II biosafety cabinet. We will collect six samples from the 1) airlock vestibule, 2) airlock thermal cover, 3) a gap in the micrometeorite shielding near the airlock, 4) a handrail near the airlock, 5) the CDRA (Carbon Dioxide Removal Assembly) vent, and 6) the VES (Vacuum Exhaust System) vent. The remaining two swabs will be reserved as controls. One swab will be exposed during the EVA without touching any surfaces to act as a blank. The final swab will remain sealed until the entire sampling kit is returned to earth. Based on previously published results from the Russian segment, we hypothesize that there will be detectable microbes at some or all of these locations. Ground-based testing of this sampling caddy confirms that the swabs remain sterile as the canisters transition in and out of vacuum. We were able to retrieve, viable bacterial and fungal cells as well as DNA from samples collected from US space suits during vacuum chamber tests lasting as long as seven hours. Based on these results and feedback from the test subjects the sampling caddy was modified to improve ergonomics and meet US EVA safety requirements. Bayonet probes were added to the sides of the sample kit as alternate mounting points. Additional locking features were added to the end effector and the filter stack to prevent inadvertent release during use. The opening mechanism was changed from one where the end effector was rocked laterally to defeat a ball detent to a twist-to-open threaded closure for similar reasons. Demonstrating, this sampling caddy’s effectiveness during a US EVA will allow us to address knowledge gaps identified in COSPAR reports and begin to define planetary protection requirements for life support systems on crewed missions to mars. This kit could also be used to collect contamination control samples during Artemis missions to verify requirements and could be easily modified for robotic sample collection.

Planetary Protection↗

NASA NTRS2014

Does Simulated Weightlessness Alter the Methylation Status of Gene Promoters During Bone Remodeling?

The skeleton interacts with its environment in a way that resembles a mechanostat - through a controlled process of bone remodeling, namely local formation and resorption, to maintain a healthy structure. During weightlessness, astronauts lose structure in weight-bearing bones due to decreased formation by osteoblasts and increased resorption by osteoclasts. In contrast, increased mechanical loading through exercise targets bone remodeling to remove and repair microdamage, improving structural integrity. In fact, recent advances in astronaut exercise regimens have prevented the deleterious changes in skeletal structure during spaceflight. However, knowledge of the molecular underpinnings of the skeletal response to spaceflight and to mechanical stimulation is limited. We propose that epigenetic modification, specifically DNA methylation, may influence osteoblast differentiation and activity during spaceflight and exercise. We hypothesize that simulated weightlessness hypermethylates pro-osteoblastogenic gene promoters and decreases expression of osteoblastogenic genes. Oppositely, we hypothesize that mechanical loading hypomethylates pro-osteoblastogenic gene promoters and increases expression of osteoblastogenic genes.

Stevick, Rebecca J.↗

NASA NTRS2017

Novel Approach to Quantification of Telomere Length with Direct Nanopore Sequencing and PCR Amplification

The ends of human chromosomes contain telomeres, or tandem arrays of repeating DNA sequences capped by multiple associated proteins that protect chromosomal ends from degradation. Telomeres function to preserve genomic stability by preventing natural chromosomal ends from being recognized as broken DNA double-strand breaks and triggering inappropriate DNA damage responses. Mounting evidence shows telomere length is an inherited trait that decreases with cellular division and normal aging. In addition, telomere length also appears to be influenced by other factors such as cellular oxidative stress, radiation and mechanical unloading of tissues as in microgravity. To measure these potential effects of the space environment on telomere lengths and cellular aging and regenerative potential we developed a novel telomere measurement approach based on nanopore sequencing of PCR amplified bar-coded chromosome termini. Specifically, telomeres can be directly enriched using barcode sequences ligated to the end of a free end- repaired telomere using the WetLab-2 facility SmartCycler on ISS. Prior to the ligation and amplification protocol a proteinase K digestion of capping proteins followed by a single 95-degree C heat denaturation of the protease is included. After digestion and bar-code ligation, PCR amplification will initiate with the ligated barcoded sequence, suppressing amplification of intra-genomic fragments and resulting in long read barcoded telomere amplicons including the nanopore motor protein sequences. Purified PCR amplicons are then used for nanopore sequencing library generation by simple addition of motor proteins and sequencing library is loaded into the MinION nanopore DNA-sequencer. Amplicon sequence reads from the nanopore device can be base-called quickly on ISS due to barcoding ligation and subsequent PCR amplification enhancing the telomere sequence resolution. If successfully implemented on ISS this technique will provide a novel means of measuring regenerative ability of somatic stem cells in astronauts, and of determining whether spaceflight in microgravity alters their telomere lengths and causes premature cellular aging.

Ma, Kristin R.↗

NASA NTRS1994

Heavy ion induced DNA-DSB in yeast and mammalian cells

Molecular changes at the DNA are assumed to be the main cause for radiation effects in a number of organisms. During the course of the last decades techniques have been developed for measuring DNA double-strand breaks (dsb), generally assumed to be the most critical DNA lesions. The outcome of all those different approaches portrays a collection of data useful for a theoretical description of radiation action mechanisms. However, in the case of heavy ion induced DNA dsb the picture is not quite clear yet and further projects and strategies have to be developed. The biological systems studied in our group are yeast and mammalian cells. While in the case of yeast cells technical and methodical reasons highlight these organisms mammalian cells reach greater importance when dsb repair studies are performed. In both types of organisms the technique of pulsed-field gel electrophoresis (PFGE) is applied, although with different modifications and evaluation procedures mainly due to the different genome sizes.

Loebrich, M.↗

NASA NTRS1998

Joining of correct and incorrect DNA ends at double-strand breaks produced by high-linear energy transfer radiation in human fibroblasts

DNA double-strand breaks (DSBs) were measured within a 3.2-Mbp NotI fragment on chromosome 21 of cells of a normal human fibroblast cell line. Correct rejoining of DSBs was followed by measuring reconstitution of the original-size NotI fragment, and this was compared to total rejoining as measured by a conventional pulsed-field gel electrophoresis technique (FAR assay). After 80 Gy of particle irradiations with LETs in the range of 7-150 keV/microm, it was found that the repair kinetics was generally slower after irradiation with high-LET particles compared to X irradiation and that a larger proportion of the breaks remained unrepaired after 24 h. On the other hand, the misrejoining frequency as measured by the difference between correct and total rejoining after 24 h did not change with LET, but was approximately the same for all radiations at this dose, equal to 25-30% of the initial breaks. This result is discussed in relation to formation of chromosomal aberrations, deletion mutations and other biological end points.

NASA Discipline Radiation Health↗