Abstracts
Rydberg B; Holley WR; Mian IS; Chatterjee A. Chromatin conformation in living cells: support for a zig-zag model of the 30 nm chromatin fiber. Journal of Molecular Biology, 1998 Nov 20, 284(1):71-84.
A new method was used to probe the conformation of chromatin in living mammalian cells. The method employs ionizing radiation and is based on the concept that such radiation induces correlated breaks in DNA strands that are in spatial proximity. Human dermal fibroblasts in G0 phase of the cell cycle and Chinese hamster ovary cells in mitosis were irradiated by X-rays or accelerated ions. Following lysis of the cells, DNA fragments induced by correlated breaks were end-labeled and separated according to size on denaturing polyacrylamide gels. A characteristic peak was obtained for a fragment size of 78 bases, which is the size that corresponds to one turn of DNA around the nucleosome. Additional peaks between 175 and 450 bases reflect the relative position of nearest-neighbor nucleosomes. Theoretical calculations that simulate the indirect and direct effect of radiation on DNA demonstrate that the fragment size distributions are closely related to the chromatin structure model used. Comparison of the experimental data with theoretical results support a zig-zag model of the chromatin fiber rather than a simple helical model. Thus, radiation-induced damage analysis can provide information on chromatin structure in the living cell. Holley WR; Chatterjee A. Clusters of DNA induced by ionizing radiation: formation of short DNA fragments. I. Theoretical modeling. Radiation Research, 1996 Feb, 145(2):188-99.
We have developed a general theoretical model for the interaction of ionizing radiation with chromatin. Chromatin is modeled as a 30-nm-diameter solenoidal fiber comprised of 20 turns of nucleosomes, 6 nucleosomes per turn. Charged-particle tracks are modeled by partitioning the energy deposition between primary track core, resulting from glancing collisions with 100 eV or less per event, and delta rays due to knock-on collisions involving energy transfers >100 eV. A Monte Carlo simulation incorporates damages due to the following molecular mechanisms: (1) ionization of water molecules leading to the formation of OH, H, eaq, etc.; (2) OH attack on sugar molecules leading to strand breaks: (3) OH attack on bases; (4) direct ionization of the sugar molecules leading to strand breaks; (5) direct ionization of the bases. Our calculations predict significant clustering of damage both locally, over regions up to 40 bp and over regions extending to several kilobase pairs. A characteristic feature of the regional damage predicted by our model is the production of short fragments of DNA associated with multiple nearby strand breaks. The shapes of the spectra of DNA fragment lengths depend on the symmetries or approximate symmetries of the chromatin structure. Such fragments have subsequently been detected experimentally and are reported in an accompanying paper (B. Rydberg, Radiat, Res. 145, 200-209, 1996) after exposure to both high- and low-LET radiation. The overall measured yields agree well quantitatively with the theoretical predictions. Our theoretical results predict the existence of a strong peak at about 85 bp, which represents the revolution period about the nucleosome. Other peaks at multiples of about 1,000 bp correspond to the periodicity of the particular solenoid model of chromatin used in these calculations. Theoretical results in combination with experimental data on fragmentation spectra may help determine the consensus or average structure of the chromatin fibers in mammalian DNA Henle ES; Roots R; Holley WR; Chatterjee A. DNA strand breakage is correlated with unaltered base release after gamma irradiation. Radiation Research, 1995 Aug, 143(2):144-50.
Unaltered base release is correlated with strand breakage for gamma-irradiated bacteriophage PM2 DNA in aqueous solution at pH 7.4. The yield of DNA strand breaks is determined by the agarose gel electrophoresis method. High-performance liquid chromatography (HPLC) is used to assay the release of unaltered nucleic bases. Previously reported HPLC methods have been updated. Unaltered base release is linear with dose up to 424 Gy, where up to 0.2% of all DNA bases are released. No detectable amounts of unaltered nucleosides are released and, besides unaltered bases, only one other product released from DNA is observed. Base release yields do not reflect the PM2 GC content of 43%. Only 76% of all prompt strand breaks appear to be associated with the release of an unaltered free base, whereby the guanine, cytosine, adenine and thymine yields are 9, 27, 18 and 22% of the prompt strand break yield, respectively. Postirradiation incubation at 37 degrees C for 24 h increases the strand break yield 1.38-fold and the unaltered base release yield 1.76-fold such that 97% of the final strand breaks appear to be associated with the release of an unaltered base, whereby the guanine, cytosine, adenine and thymine yields are 10, 36, 23 and 28% of the final strand break yield, respectively. These data indicate that, given proper conditions, nearly every strand break leads to a base release. The bearing of these results on OH radical attack leading to strand breakage and base release is discussed. Chatterjee A; Schmidt JB; Holley WR. Monte Carlo approach in assessing damage in higher order structures of DNA. Basic Life Sciences, 1994, 63:225-35; discussion 235-41.
We have developed a computer monitor of nuclear DNA in the form of chromatin fibre. The fibres are modeled as a ideal solenoid consisting of twenty helical turns with six nucleosomes per turn. The chromatin model, in combination with are Monte Carlo theory of radiation damage induces by charged particles, based on general features of tack structure and stopping power theory, has been used to evaluate the influence of DNA structure on initial damage. An interesting has emerged from our calculations. Our calculated results predict the existence of strong spatial correlations in damage sites associated with the symmetries in the solenoidal model. We have calculated spectra of short fragments of double stranded DNA produced by multiple double strand breaks induced by both high and low LET radiation. The spectra exhibit peaks at multiples of approximately 85 base pairs (the nucleosome periodicity), and approximately 1000 base pairs (solenoid periodicity). Preliminary experiments to investigate the fragment distributions from irradiated DNA, made by B. Rydberg at Lawrence Berkeley Laboratory, confirm the existence of short DNA fragments and are in substantial agreement with the predictions of our theory. Holley WR; Chatterjee A. A computational approach to the relationship between radiation induced double strand breaks and translocations. Basic Life Sciences, 1994, 63:251-8; discussion 258-9.
A theoretical framework is presented which provides a quantitative analysis of radiation induced translocations between the ab1 oncogene on CH9q34 and a breakpoint cluster region, bcr, on CH 22q11. Such translocations are associated frequently with chronic myelogenous leukemia. The theory is based on the assumption that incorrect or unfaithful rejoining of initial double strand breaks produced concurrently within the 200 kbp intron region upstream of the second abl exon, and the 16.5 kbp region between bcr exon 2 and exon 6 interact with each other, resulting in a fusion gene. for an x-ray dose of 100 Gy, there is good agreement between the theoretical estimate and the one available experimental result. The theory has been extended to provide dose response curves for these types of translocations. These curves are quadratic at low doses and become linear at high doses. Roots, R; Henle, E; Holley, W R; Chatterjee, A. Measurements of nucleic bases released after gamma irradiation of DNA in solution in air. Radiation Research, v.125, n.3, (1991): 288-292.
The release of unaltered nucleic bases from .gamma.-irradiated DNA in a dilute buffered aqueous solution was studied in both salmon sperm and superhelical viral DNA. Analyses of freed bases were made by high-performance liquid chromatography. An elution protocol was developed for maximum separation of the four nucleic bases and nucleosides with a sensitivity of 10-20 pmol of nucleic base. It was found that: (i) both prompt and delayed release of bases postirradiation occur in both types of DNA; (ii) these yields (G-values) were measured to be 10-15 times higher for the salmon sperm DNA in comparison to the SV40 DNA; (iii) the A-T/G-C ratio in the DNA was not reflected in the ratios of the released base; and (iv) based on measurements made by use of DNA strand breaks in SV40 DNA (unpublished results), less than half of all breaks result in the release of an undamaged base. Roots, R; Holley, W; Chatterjee, A; Irizarry, M; Kraft, G. The formation of strand breaks in DNA after high-LET irradiation: A comparison of data from in vitro and cellular systems. International Journal of Radiation Biology, v.58, n.1, (1990): 55-70.
This paper presents a summary of our understanding to date of the formation of DNA strand breaks induced by highly energetic particle beams (high-LET radiation). We have compared our own recent data on the formation of strand breaks induced in DNA in an aqueous solution with our previous data and those of others available from the literature for similar lesions made in cellular DNA. When the strand break induction frequency, as number of breaks per Gy per unit DNA, is plotted against LET, a series of biological effect curves (one for each particle atomic number Z) is obtained. The frequency of the formation of single-strand breaks has an RBE of less than 1 for DNA in solution and for DNA in the cell; the frequency of the formation of double-strand breaks (dsb) also has an RBE of less than 1 for DNA in a solution containing low amounts of free radical scavenger(s), while the RBE can be greater than 1 in the 50-200 keV/.mu.m range for cellular DNA. RBE values are with respect to X-rays or cobalt .gamma.-rays. In cells the level of unrejoined strand breaks is also highest in the 50-200 keV/.mu.m range and may reach 25-35% of the initial break yield depending on particle energy and Z-value. These irreparable lesions include double-strand scissions and some form(s) of single-strand breaks. The data presented cover results obtained for helium to uranium particles, with an LET range of 16 to 160,000 keV/.mu.m. When different biological end-points are compared a strong correlation is found between the induction of dsb, chromosomal abnormalities and mutation induction. Holley, W R; Chatterjee, A; Magee, J L. Production of DNA strand breaks by direct effects of heavy charged particles. Radiation Research, v.121, n.2, (1990): 161-168.
A theoretical model has been developed to calculate the yields of single- and double-strand breaks in DNA induced by direct effects of ionizing radiation. In this model, which involves no fitted parameters, elements of track structure and stopping power theory are combined with a detailed geometrical description of DNA to calculate the energy deposited by fast charged particles to DNA molecules. The average energy per interaction with a DNA molecule is estimated to be 30 eV from the available data on oscillator strength measurements. These ideas have been incorporated in a Monte Carlo computer program using Poisson statistics to treat the stochastic nature of the energy deposition processes and thereby determine the excitation and ionization states of the molecule. Each ionization reaction on the DNA backbone is assumed to lead to a DNA strand break. In our model double-strand breaks result from nearby independent breaks on opposite strands. Our calculated single- and double-strand break yields compare well with measured cellular data under conditions such that direct effects are thought to dominate strand break production.