Ionizing radiation-induced fragmentation of plasmid DNA – Atomic force microscopy and biophysical modeling

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Abstract

It is widely accepted that DNA double-strand breaks (DSBs) are closely correlated with radiation-induced cell killing and are the most critical lesions related to cellular endpoints like mutagenesis and transformation. High linear energy transfer (LET) radiation produces more severe and complex damage due to the fact that induced DSBs are not randomly distributed but clustered at different levels of DNA organization. In this paper, direct visualization of DSBs induced in a plasmid supercoiled DNA by low- and high-LET radiation is presented. Resulting DNA fragments distributions obtained by use of atomic force microscopy (AFM) are shown. Moreover, a biophysical model of spatially correlated DSBs formation in the framework of the Local Effect Model (LEM) is introduced and its predictions on DNA fragment formation are discussed.

Introduction

Ionizing radiation is known to cause many different types of lesions in DNA, most importantly base damage and strand breaks. Among them the double-strand breaks (DSBs) are considered to be most severe. There is experimental evidence suggesting that both, their initial level and residual/unrepaired level, can be correlated with the various biological end-points like cell death, loss of reproductive capacity, mutations or carcinogenesis (Alpen, 1998, vonSonntag, 1987). DSB production is expected to be determined by both, the physical properties of the radiation (i.e. the spatial distribution of ionization events) and the chemical environment of the DNA. Both issues can be conveniently addressed by studying radiation damage in viral or plasmid DNA as very simple experimental model systems. Different studies using heavy ion irradiation were performed in highly protective solutions (Brons et al., 2001, Christensen et al., 1972, Taucher-Scholz et al., 1992) as well as at low scavenging concentrations (Roots et al., 1990, Taucher-Scholz and Kraft, 1999). In contrast to X-rays, high-LET radiation features a highly structured deposition of energy along the track of the charged particles (Krämer and Kraft, 1994). Because of the nature of energy deposition (ionization events), high-LET radiation is expected to induce more complex and severe lesions correlated along individual particle tracks (Goodhead, 1989, Krämer and Kraft, 1994, Nikjoo et al., 1999). As a consequence, a high density of biological damage is induced at the sites close to the particle trajectory. This localization effect is responsible for damage clustering. So far, clustering of DNA lesions has been investigated by incubation of irradiated DNA with base excision repair enzymes (Gulston et al., 2002, Milligan et al., 2001, Sutherland et al., 2001). The results have shown that indeed additional DSBs are revealed, indicating association of the radiation-induced DSBs with nearby base damages.

From the theoretical point of view, a detailed calculation of the track structure reflecting the spatial distribution of ionizations (Kraft et al., 1992) should allow to estimate the yields of DSBs and additionally can contribute to the further elucidation of the radiation-quality dependence. Therefore, parallel to the experimental effort, many Monte Carlo studies were performed to simulate the interaction of ionizing radiation with DNA in various conformations including higher levels of chromatin organization in mammalian cells (Höglund et al., 2000, Holley and Chatterjee, 1996, Nikjoo et al., 1999, Nikjoo et al., 2001, Watanabe and Saito, 2002).

Experimentally, the differences in DSB induction after low- and high-LET radiation can be investigated by fragmentation analysis using simple in vitro models, i.e. plasmid DNA. Commonly available experimental techniques to quantify strand breaks in plasmids rely on gel electrophoresis (Milligan et al., 1993, Roots et al., 1989, Taucher-Scholz and Kraft, 1999). Although these methods provide information on DNA DSBs resulting from the irradiation, they are limited by their inability to resolve individual DNA fragments within smeared distributions of fragments induced by radiation. In particular, short fragments are difficult to be detected. This limitation is also apparent in experimental studies addressing the fragmentation of cellular DNA (see (Prise et al., 1998) for review).

Complementary to these indirect methods, DNA fragmentation can be directly quantified using atomic force microscopy (AFM). Since their invention (Binnig et al., 1986) AFM has been increasingly used in biological science (Hansma and Hoh, 1994) combining the high spatial resolution (allowing direct visualization of biomolecules) with the ability to image biological samples under their physiological environment. The powerful imaging capability of AFM has also expanded into radiobiological research (Pang et al., 1997). Using AFM, the plasmid DNA was used to investigate the DSBs induced by X-rays, neutrons, electrons and alpha particles (Boichot et al., 2002, Pang et al., 1998, Pang et al., 2005). First studies regarding the DNA damage induced in plasmid DNA by heavy ions were also reported (Psonka et al., 2005).

Here, we report on the investigation of ionizing radiation induced DSBs in plasmid DNA directly visualized by AFM. We present measured DNA fragment distributions after irradiation with X-rays and 3.9 MeV u−1 Ni ions. Furthermore, we compare the experimentally obtained fragmentation patterns with the theoretical predictions based on the Local Effect Model (LEM).

Section snippets

Plasmid DNA

Double-stranded supercoiled ΦX174 plasmid DNA (about 82% of supercoiled based on electrophoretic analysis, Promega, Madison WI) was used for the experiment. Mock-irradiated control samples analyzed by means of standard electrophoresis showed that 77% and 72% of the DNA was supercoiled for sample sets irradiated with X-rays or Ni ions, respectively. Both controls showed less than 1% of linear form molecules. The plasmid has 5386 bp which corresponds to approximately 1830 nm (assuming 0.34 nm bp−1

Theoretical framework of the Local Effect Model

The Local Effect Model (LEM) is a semi-empirical model originally developed within the tumor therapy project at GSI to predict cell inactivation probabilities by heavy ions (Scholz and Kraft, 1996, Scholz and Kraft, 2004). The model can also be used to calculate DSB induction in DNA (Brons et al., 2003). The calculations are based on the pattern of local energy deposition and rely on the following informations:

  • the size of a critical target, which in this case is the plasmid DNA molecule,

  • the

Results

Fig. 1 shows some exemplary topographies of the DNA irradiated with X-rays or Ni ions at 250 Gy, as imaged by AFM. For each dose and radiation quality, the recorded AFM images were analyzed. The type of the induced damage was identified according to the conformation of the detected DNA molecules using defined differentiation criteria. Both intact supercoiled and circular relaxed molecules were detected. Molecules containing double-strand breaks were identified as linear ones. For each observed

Discussion

In this paper, the fragmentation profiles of plasmid DNA irradiated with X-rays and particles (3.9 MeV u−1 Ni ions) were shown and compared. The differences in fragment distributions were attributed to the local deposition of energy by ionization events. Single particle tracks result in spatially correlated ionization events that in turn are responsible for the pattern of DSB distribution. Spatially correlated DSBs within single particle tracks lead to the induction of complexes of locally

Acknowledgments

The authors acknowledge the financial and logistic support by Prof. G. Kraft and Prof. R. Neumann from the GSI biophysics and material science research departments. This research project is supported in part by KBN Grant 1P03B 087 30 (K. Psonka and E. Gudowska-Nowak), Marie Curie COCOS TOK Programme (E. Gudowska-Nowak) and co-financed from the European Social Fund and national budget in the frame of the Integrated Regional Operational Programme.

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