РАДИАЦИОННАЯ БИОЛОГИЯ. РАДИОЭКОЛОГИЯ, 2007, том 47, № 3, с. 292-296
PROCEEDINGS OF THE 4TH INTERNATIONAL WORKSHOP ON SPACE RADIATION RESEARCH — AND 17TH ANNUAL NASA SPACE RADIATION HEALTH -
INVESTIGATORS' WORKSHOP (Moscow-St.-Petersburg, June 5-9, 2006)
УДК [577.2:539.1.04]+577.346
Biophysical Modelling of DNA DSB Repair Following High LET Irradiation
© 2007 I. V. Salnikov, Yu. A. Eidelman, S. G. Andreev*
Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia
One of the quantitative methods used in DNA repair research is a measurement of the size-distribution of DNA fragments at different times following cell irradiation. The aim of the present study was to evaluate the relationship between the experimentally observed size-distributions of DNA fragments and the parameters of doublestrand break (DSB) repair. A biophysical model of DNA DSB repair in chromosomal DNA including DSB clusters repair was proposed. Complex shapes of (1) DNA fragments distribution at different repair times, (2) rejoining kinetics for DNA fragments in different length intervals, (3) total fragments rejoining kinetics were simultaneously described with rates of DSB repair different for active/inactive chromatin compartments.
High LET radiation, repair, DNA doublestrand breaks, modelling, track structure.
One of the experimental methods developed for double-strand break (DSB) repair study is based on DNA fragment scoring at different times following cell irradiation using pulse field gel electrophoresis (PFGE) [1]. Understanding of the shape of DNA DSB fragment rejoining curves in wide range of length intervals together with fragments distribution for different repair times might provide deeper insight into quantitative relationships between non-random distribution of initial DNA DSB or DSB clusters in cellular DNA [1, 2] and their repair.
Mechanistic modelling may serve as an effective tool for testing various hypotheses about the DNA breakage and DSB rejoining mechanisms. A phenome-nological modelling approach has been proposed to explain quantitatively the experimental data on DSB rejoining kinetics for low and high LET radiation [3]. However, that approach has not taken into account several important mechanistic aspects such as structure of charged particle tracks, DNA organisation in chromosomes, etc. Another technique [4] has dealt with radiation-induced DNA fragments induction taking into account track structure. However, there has been no mechanistic modelling of DNA package on chromosome level. Moreover, work [4] has not aimed at DSB repair study.
We have developed a new method quantifying induction of DSB and DSB clusters (multiple DSB formed by multiple track crossings with DNA within the same chromosome) which are manifested as heterogeneous, or non-random DNA fragments distribution
* Corresponding address: Institute of Biochemical Physics, Kosy-gin str., 4, Moscow, 119991 Russia; Phone: +7 (495) 939-71-94; fax: +7 (495) 137-41-01; e-mail: andreev_sg@mail.ru.
[2, 5, 6]. That method provided mechanistic data-based simulation of DNA package in interphase chromosomes and incorporated information about charged particle track structure into DNA breakage modelling [2, 5, 6]. Unlike previous approaches [3, 4, 7] we have modelled the globular structure of interphase chromosomes by taking into account realistic chromatid chain package formed by volumetric and excluded volume interactions (self-avoiding chain). It has been shown that the chromosome structure simulated was compatible with experimental data on DNA folding in interphase chromosome [2, 5, 6]. Modelling techniques developed [2, 6] significantly decreased uncertainties of prediction of DNA fragment distributions where chromosome structure simulation is one of the key problems.
In the present work the computational approach for prediction of DNA fragment distribution [2] was extended to quantify DSB repair observed by PFGE based fragment analysis technique [1, 8]. The main goal of the present work was a theoretical study of repair of DSB clusters in chromosomal DNA. The specific aim was to predict DNA fragment size distributions at different times following high-LET irradiation and to determine parameters of DSB repair from PFGE data analysis. We provide a modelling evidence that DSB clusters formed in inactive chromatin are repaired slower than those in active chromatin. Alternative mechanisms, in particular, slow DSB repair rate being due to DSB complexity at molecular level [9, 10], need to be tested but are outside the scope of this paper.
METHODS
Stochastic track structure of different LET ions modelled by Monte Carlo methods on the event-by-event
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Fig. 1. Fragment-size distributions at different repair times: A - t = 0 h; B - t = 0.5 h, C - t = 3 h, D - t = 6 h. Nitrogen ions, LET 125 keV/|J,m, dose 100 Gy. 1 - data [1], human fibroblasts; 2 - simulation.
basis was described elsewhere [2]. The developed DNA target model includes six levels of DNA organisation: from double helix through chromatin fibre loops to the whole human interphase chromosome [11]. The 30 nm chromatin fibre was simulated as a selfavoiding chain composed of 5 kbp segments [12]. The structure of a human interphase chromosome was modelled, taking into account active and inactive chromosome compartments. Active regions were simulated as rosettelike structures (superdomains, SDs) consisting of chromatin loops with the mean DNA content about 100 kbp attached to the protein core [2, 6, 12]. Inactive regions were represented by non-looped 30 nm chromatin fibre folded in blobs between SDs. An interphase chromosome was represented as a flexible chain of SDs and blobs [6]. The organisation of the chromosome within a cell nucleus was modelled as further folding of the chain of subunits (SDs and blobs) due to volumetric and excluded volume interactions between them [6].
The superimposing of chromosome structure and each stochastic particle track allowed calculation of energy deposition in every nucleotide of the DNA molecule. The input data for the DNA-damage computer code comprised coordinates, transferred energies and types of interaction [2]. The volumetric DNA strand breakage model was used to calculate single-strand break (SSB) and DSB frequencies [2, 13-15]. Size spectra of double-stranded DNA fragments were obtained according to DSB-DSB fragment scoring [5] for
the structure of human interphase chromosome 1 as simulated previously [6]. A method has been developed to predict the size distribution of DNA fragments formed by both radiation-induced and background DSB for any LET and non-random background damage [5]. In the present work that method was extended to investigate DSB repair.
DSB repair was studied by the Monte Carlo technique. A DNA fragment size scored at any repair time t was defined as the number of DNA base pairs between two neighboring DSBs existing at that time. As we used in the present work a detailed model of chromosome structure, taking into account difference in the structure of active-inactive compartments, it seemed reasonable to introduce in general case a difference of DNA repair rate in these compartments. We proposed that the repair rate of each DSB is independent of dose, time, LET, but is dependent on DSB position in the active or inactive chromosome compartments.
The number of DNA fragments for different size-intervals was calculated at different repair times. The DSB repair rates in active and inactive chromatin were determined by fitting the experimental data on fragment number in several size ranges measured at different repair times in human fibroblasts after irradiation with nitrogen ions, LET = 125 keV/^m [18.].
Repair time, h
Fig. 2. Fragment repair kinetics depending on fragment length: A - 5-97 kbp length range, B - 97-375 kbp, C - 375-930 kbp, D -930-3500 kbp, E - 3500-5700 kbp, F - all fragments (5-5700 kbp).
Nitrogen ions, LET 125 keV/^m, dose 100 Gy. 1 - data [2], human fibroblasts; 2 - simulation.
RESULTS
The computed and measured fragment-size distributions are presented in Fig. 1. The distributions were calculated for DSB induction (Fig. 1, panel A) and for different repair times (Fig. 1, panels B-D). The obtained distributions are in good agreement with the experimental data [1]. The simulations were made for repair rates varying for different chromosomal compartments, i.e. for active vs inactive chromatin. The rates of DSB repair were determined from the best fit of calculated and observed curves and were equal 1.9 h-1 for active and 0.12 h-1 for inactive chromatin.
Fig. 2 (panels A-E) presents the simulated kinetics of DNA fragment rejoining for different size intervals compared with the experimental data [8]. Rejoining kinetics for the total number of fragments (in the range 55700 kbp obtained by fragment analysis) is presented in Fig. 2, panel F. The simulation results also agree well with the experimental data both for all fragment-size intervals and for total number of fragments except for the region 3500-5700 kbp (Fig. 2, panel E).
DISCUSSION
The most appropriate data for DSB quantitative analysis are DNA fragment distributions measured by PFGE. It has been shown that correct evaluation of fast and slow components of DSB rejoining kinetics curves
requires counting of DNA fragments in different length intervals [1]. As most experimental studies of DNA breakage by X-rays have been carried out by FAR technique, i.e. without fragment analysis (see for example [1, 18, 19]), we focused our study on high-LET induced DNA fragmentation. Experimental data for nitrogen ions were analysed by the present technique based on mechanistic modelling of chromosome organization and DNA breakage by taking into accoun
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