Spectrum of complex DNA damages depends on the incident radiation.
ABSTRACT Ionizing radiation induces bistranded clustered damages--two or more abasic sites, oxidized bases and strand breaks on opposite DNA strands within a few helical turns. Since clusters are refractory to repair and are potential sources of double-strand breaks (DSBs), they are potentially lethal and mutagenic. Although induction of single-strand breaks (SSBs) and isolated lesions has been studied extensively, little is known about the factors affecting induction of clusters other than DSBs. To determine whether the type of incident radiation could affect the yields or spectra of specific clusters, we irradiated genomic T7 DNA, a simple 40-kbp linear, blunt-ended molecule, with ion beams [iron (970 MeV/nucleon), carbon (293 MeV/nucleon), titanium (980 MeV/nucleon), silicon (586 MeV/nucleon), protons (1 GeV/nucleon)] or 100 kVp X rays and then quantified DSBs, Fpg-oxypurine clusters and Nfo-abasic clusters using gel electrophoresis, electronic imaging and number average length analysis. The yields (damages/Mbp Gy(-1)) of all damages decreased with increasing linear energy transfer (LET) of the radiation. The relative frequencies of DSBs compared to abasic and oxybase clusters were higher for the charged particles-including the high-energy, low-LET protons-than for the ionizing photons.
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ABSTRACT: Abstract Background: Ionizing radiation induces a plethora of DNA damages including double strand breaks (DSB) that may trigger a series of events such as transcription, DNA repair and alteration in the conformation of chromatin structure in human cells. We have made an attempt to study the conformational changes in chromatin fibers in irradiated Human Peripheral Blood Mononuclear cells (PBMC) using Dynamic Light Scattering (DLS) as a new tool. Materials and Method: Venous blood samples were collected from 10 random, healthy individuals with written informed consent, approved by institutional ethics committee. PBMC were separated from blood, irradiated with different doses of gamma radiation between 0.25 Gy to 1.0 Gy. Native chromatin was isolated from irradiated PBMC and changes in the hydrodynamic diameter of the chromatin fiber were measured using DLS. Both dose response and time kinetics was studied in order to see the chromatin changes. Radiation induced DNA double strand breaks were measured using gamma-H2AX (histone 2A member X) as a biomarker using flow cytometry and foci were visualized in confocal microscopy. Results: A significant alteration in hydrodynamic diameter of the chromatin fiber was observed at lower doses (0.25 and 0.50 Gy), whereas at higher dose (1.0 Gy), the size of the chromatin fiber was comparable to unirradiated control. Among the 10 individuals studied, five individuals showed significant increase (P ≤ 0.002) in hydrodynamic size at 0.25 Gy whereas four individuals showed significant decrease (P ≤ 0.009) at 0.25 Gy. One individual did not show any significant difference as compared to control. However, dose dependent increase in gamma-H2AX fluorescence signals as well as foci number was observed. Increased fragmentation of chromatin fiber was also observed using Atomic Force Microscopy at higher doses. Conclusion: Radiation-induced DNA damage response can lead to individual specific conformational changes in chromatin structure at lower doses (0.25 Gy and 0.50 Gy) which can be detected using dynamic light scattering method in resting human PBMC.International journal of radiation biology. 05/2014;
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ABSTRACT: The primary structure of Histone Acetyltransferase 1 (Hat1) has been conserved throughout evolution; however, despite its ubiquity, its cellular function is not well characterized. To study its in vivo acetylation pattern and function, we utilized shRNAmir against Hat1 expressed in the well-substantiated HeLa (human cervical cancer) cell line. To reduce the interference by enzymes with similar HAT specificity, we used HeLa cells expressing histone acetyltransferase Tip60 with mutated acetyl-CoA binding site that abrogates its enzyme activity (mutant HeLa-tip60). Two shRNAmir were identified that reduced the expression of the cytoplasmic and nuclear forms of Hat1. Cytosolic protein preparations from these two clones showed decreased levels of acetylation of lysine 5 (K5) and K12 on histone H4, with the concomitant loss of the acetylation of histone H2A at K5. This pattern of decreased acetylation of H2AK5 was well defined in preparations of histone protein and insoluble nuclear-protein (INP) fractions as well. Abrogating the Hat1 expression caused a 74 % decrease in colony-forming efficiency of mutant HeLa-tip60 cells, reduced the size of the colonies by 50 %, and decreased the amounts of proteins with molecular weights below 35 kDa in the INP fractions.Molecular and Cellular Biochemistry 03/2014; · 2.33 Impact Factor
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ABSTRACT: DNA methylation is an epigenetic mechanism that drives phenotype and that can be altered by environmental exposures including radiation. The majority of human radiation exposures occur in a relatively low dose range; however, the biological response to low dose radiation is poorly understood. Based on previous observations, we hypothesized that in vivo changes in DNA methylation would be observed in mice following exposure to doses of high linear energy transfer (LET) (56) Fe ion radiation between 10 and 100 cGy. We evaluated the DNA methylation status of genes for which expression can be regulated by methylation and that play significant roles in radiation responses or carcinogenic processes including apoptosis, metastasis, cell cycle regulation, and DNA repair (DAPK1, EVL, 14.3.3, p16, MGMT, and IGFBP3). We also evaluated DNA methylation of repeat elements in the genome that are typically highly methylated. No changes in liver DNA methylation were observed. Although no change in DNA methylation was observed for the repeat elements in the lungs of these same mice, significant changes were observed for the genes of interest as a direct effect and a delayed effect of irradiation 1, 7, 30, and 120 days post exposure. At delayed times, differences in methylation profiles among genes were observed. DNA methylation profiles also significantly differed based on dose, with the lowest dose frequently affecting the largest change. The results of this study are the first to demonstrate in vivo high LET radiation-induced changes in DNA methylation that are tissue and locus specific, and dose and time dependent. Environ. Mol. Mutagen. 55:266-277, 2014. © 2013 Wiley Periodicals, Inc.Environmental and Molecular Mutagenesis 04/2014; 55(3):266-77. · 3.71 Impact Factor