William R. Holley

Clusters of DNA damage induced by ionizing radiation : formation of short DNA fragments. I: Theoretical modeling

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.

Production of DNA strand breaks by direct effects of heavy charged particles

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.

A model for interphase chromosomes and evaluation of radiation-induced aberrations

Abstract Holley, W. R., Mian, I. S., Park, S. J., Rydberg, B. and Chatterjee, A. A Model for Interphase Chromosomes and Evaluation of Radiation-Induced Aberrations. Radiat. Res. 158, 568–580 (2002). We have developed a theoretical model for evaluating radiation-induced chromosomal exchanges by explicitly taking into account interphase (G0/G1) chromosome structure, nuclear organization of chromosomes, the production of double-strand breaks (DSBs), and the subsequent rejoinings in a faithful or unfaithful manner. Each of the 46 chromosomes for human lymphocytes (40 chromosomes for mouse lymphocytes) is modeled as a random polymer inside a spherical volume. The chromosome spheres are packed randomly inside a spherical nucleus with an allowed overlap controlled by a parameter Ω. The rejoining of DSBs is determined by a Monte Carlo procedure using a Gaussian proximity function with an interaction range parameter σ. Values of Ω and σ have been found which yield calculated results of interchromosomal aberration frequencies that agree with a wide range of experimental data. Our preferred solution is one with an interaction range of 0.5 μm coupled with a relatively small overlap parameter of 0.675 μm, which more or less confirms previous estimates. We have used our model with these parameter values and with resolution or detectability limits to calculate yields of translocations and dicentrics for human lymphocytes exposed to low-LET radiation that agree with experiments in the dose range 0.09 to 4 Gy. Five different experimental data sets have been compared with the theoretical results. Essentially all of the experimental data fall between theoretical curves corresponding to resolution limits of 1 Mbp and 20 Mbp, which may reflect the fact that different investigators use different limits for sensitivity or detectability. Translocation yields for mouse lymphocytes have also been calculated and are in good agreement with experimental data from 1 cGy to 10 cGy. There is also good agreement with recent data on complex aberrations. Our model is expected to be applicable to both low- and high-LET radiation, and we include a sample prediction of the yield of interchromosomal rejoining in the dose range 0.22 Gy to 2 Gy of 1000 MeV/nucleon iron particles. This dose range corresponds to average particle traversals per nucleus ranging from 1.0 to 9.12.

DNA strand breakage is correlated with unaltered base release after gamma irradiation

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.

Dose-Dependent Misrejoining of Radiation-Induced DNA Double-Strand Breaks in Human Fibroblasts: Experimental and Theoretical Study for High- and Low-LET Radiation

Abstract Rydberg, B., Cooper, B., Cooper, P. K., Holley, W. R. and Chatterjee, A. Dose-Dependent Misrejoining of Radiation-Induced DNA Double-Strand Breaks in Human Fibroblasts: Experimental and Theoretical Study for High- and Low-LET Radiation. Radiat. Res. 163, 526–534 (2005). Misrejoining of DNA double-strand breaks (DSBs) was measured in human primary fibroblasts after exposure to X rays and high-LET particles (helium, nitrogen and iron) in the dose range 10–80 Gy. To measure joining of wrong DNA ends, the integrity of a 3.2-Mbp restriction fragment was analyzed directly after exposure and after 16 h of repair incubation. It was found that the misrejoining frequency for X rays was nonlinearly related to dose, with less probability of misrejoining at low doses than at high doses. The dose dependence for the high-LET particles, on the other hand, was closer to being linear, with misrejoining frequencies higher than for X rays, particularly at the lower doses. These experimental results were simulated with a Monte Carlo approach that includes a cell nucleus model with all 46 chromosomes present, combined with realistic track structure simulations to calculate the geometrical positions of all DSBs induced for each dose. The model assumes that the main determinant for misrejoining probability is the distance between two simultaneously present DSBs. With a Gaussian interaction probability function with distance, it was found that the data for both low- and high-LET radiation could be fitted with an interaction distance (sigma of the Gaussian curve) of 0.25 μm. This is half the distance previously found to best fit chromosomal aberration data in human lymphocytes using the same methods (Holley et al., Radiat. Res. 158, 568–580, 2002). The discrepancy may indicate inadequacies in the chromosome model, for example insufficient chromosomal overlap, but may also be partly due to differences between fibroblasts and lymphocytes.

Spatial Distribution and Yield of DNA Double-Strand Breaks Induced by 3-7 MeV Helium Ions in Human Fibroblasts

Abstract Rydberg, B., Heilbronn, L., Holley, W. R., Löbrich, M., Zeitlin, C., Chatterjee, A. and Cooper, P. K. Spatial Distribution and Yield of DNA Double-Strand Breaks Induced by 3–7 MeV Helium Ions in Human Fibroblasts. Radiat. Res. 158, 32–42 (2002). Accelerated helium ions with mean energies at the target location of 3–7 MeV were used to simulate α-particle radiation from radon daughters. The experimental setup and calibration procedure allowed determination of the helium-ion energy distribution and dose in the nuclei of irradiated cells. Using this system, the induction of DNA double-strand breaks and their spatial distributions along DNA were studied in irradiated human fibroblasts. It was found that the apparent number of double-strand breaks as measured by a standard pulsed-field gel assay (FAR assay) decreased with increasing LET in the range 67–120 keV/μm (corresponding to the energy of 7–3 MeV). On the other hand, the generation of small and intermediate-size DNA fragments (0.1–100 kbp) increased with LET, indicating an increased intratrack long-range clustering of breaks. The fragment size distribution was measured in several size classes down to the smallest class of 0.1–2 kbp. When the clustering was taken into account, the actual number of DNA double-strand breaks (separated by at least 0.1 kbp) could be calculated and was found to be in the range 0.010–0.012 breaks/Mbp Gy–1. This is two- to threefold higher than the apparent yield obtained by the FAR assay. The measured yield of double-strand breaks as a function of LET is compared with theoretical Monte Carlo calculations that simulate the track structure of energy depositions from helium ions as they interact with the 30-nm chromatin fiber. When the calculation is performed to include fragments larger than 0.1 kbp (to correspond to the experimental measurements), there is good agreement between experiment and theory.

Quarks with Unit Charge: A Search for Anomalous Hydrogen

Quarks of charge +1 and other anomalous hydrogen have been sought by using the 88-inch cyclotron at Berkeley as a high-energy mass spectrometer, with natural hydrogen and deuterium as the sources of ions. No quarks were observed, and limits were placed on their ratio to protons on the earth that vary from < 2 x 10–19for high masses (3 to 8.2 atomic mass units) to 10–13 for the lowest masses (< ⅓ atomic mass unit).

Statistical modelling and phylogenetic analysis of a deaminase domain.

Deamination reactions are catalyzed by a variety of enzymes including those involved in nucleoside/nucleotide metabolism and cytosine to uracil (C-->U) and adenosine to inosine (A-->I) mRNA editing. The active site of the deaminase (DM) domain in these enzymes contains a conserved histidine (or rarely cysteine), two cysteines and a glutamate proposed to act as a proton shuttle during deamination. Here, a statistical model, a hidden Markov model (HMM), of the DM domain has been created which identifies currently known DM domains and suggests new DM domains in viral, bacterial and eucaryotic proteins. However, no DM domains were identified in the currently predicted proteins from the archaeon Methanococcus jannaschii and possible causes for, and a potential means to ameliorate this situation are discussed. In some of the newly identified DM domains, the glutamate is changed to a residue that could not function as a proton shuttle and in one instance (Mus musculus spermatid protein TENR) the cysteines are also changed to lysine and serine. These may be non-competent DM domains able to bind but not act upon their substrate. Phylogenetic analysis using an HMM-generated alignment of DM domains reveals three branches with clear substructure in each branch. The results suggest DM domains that are candidates for yeast, platyhelminth, plant and mammalian C-->U and A-->I mRNA editing enzymes. Some bacterial and eucaryotic DM domains form distinct branches in the phylogenetic tree suggesting the existence of common, novel substrates.

Biochemical mechanisms and clusters of damage for high-let radiation

Using mechanisms of indirect and direct radiation, a generalized theory has been developed to account for strand break yields by high-LET particles. The major assumptions of this theory are: (i) damage at deoxyribose sites results primarily in strand break formation and (2) damage to bases leads to a variety of base alterations. Results of the present theory compare well with cellular data without enzymatic repair. As an extension of this theory, we show that damage clusters are formed near each double strand break for high-LET radiation only. For 10 MeV/n (LET = 450 keV/micrometer) neon ions, the results show that on average there are approximately 3 additional breaks and approximately 3 damaged bases formed near each double strand break. For 100 MeV/n helium ions (LET = 3 keV/micrometer), less than 1% of the strand breaks have additional damage within 10 base pairs.

Measurements of nucleic bases released after gamma irradiation of DNA in solution in air.

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 us of DNA strand breaks in SV40 DNA (unpublished results), less than half of all breaks result in the release of an undamaged base.