DSB repair model for mammalian cells in early S and G1 phases of the cell cycle: application to damage induced by ionizing radiation of different quality.

The purpose of this work is to test the hypothesis that kinetics of double strand breaks (DSB) repair is governed by complexity of DSB. To test the hypothesis we used our recent published mechanistic mathematical model of DSB repair for DSB induced by selected protons, deuterons, and helium ions of different energies representing radiations of different qualities. In light of recent advances in experimental and computational techniques, the most appropriate method to study cellular responses in radiation therapy, and exposures to low doses of ionizing radiations is using mechanistic approaches. To this end, we proposed a 'bottom-up' approach to study cellular response that starts with the DNA damage. Monte Carlo track structure method was employed to simulate initial damage induced in the genomic DNA by direct and indirect effects. Among the different types of DNA damage, DSB are known to be induced in simple and complex forms. The DSB repair model in G1 and early S phases of the cell cycle was employed to calculate the repair kinetics. The model considers the repair of simple and complex DSB, and the DSB produced in the heterochromatin. The inverse sampling method was used to calculate the repair kinetics for each individual DSB. The overall repair kinetics for 500 DSB induced by single tracks of the radiation under test were compared with experimental results. The results show that the model is capable of predicting the repair kinetics for the DSB induced by radiations of different qualities within an accepted range of uncertainty.

The non-homologous end-joining (NHEJ) mathematical model for the repair of double-strand breaks: II. Application to damage induced by ultrasoft X rays and low-energy electrons.

We investigated the kinetics of simple and complex types of double-strand breaks (DSB) using our newly proposed mechanistic mathematical model for NHEJ DSB repair. For this purpose the simulated initial spectrum of DNA DSB, induced in an atomistic canonical model of B-DNA by low-energy single electron tracks, 100 eV to 4.55 keV, and the electrons generated by ultrasoft X rays (CK, AlK and TiK), were subjected to NHEJ repair processes. The activity elapsed time of sequentially independent steps of repair performed by proteins involved in NHEJ repair process were calculated for separate DSB. The repair kinetics of DSBs were computed and compared with published data on repair kinetics obtained by pulsed-field gel electrophoresis method. The comparison shows good agreement for V79-4 cells irradiated with ultrasoft X rays. The average times for the repair of simple and complex DSB confirm that double-strand break complexity is a potential explanation for the slow component of DSB repair observed in V79-4 cells irradiated by ultrasoft X rays.

Determination of DNA structural detail using radioprobing.

PURPOSE: To put radioprobing into context as a relatively new method of determining structural detail in deoxyribonucleic acid (DNA), and to review its use since first proposed in 1997. The key feature of the method is that, by experiment or simulation, a radionuclide such as iodine-125 ((125)I) is placed near the DNA at a known point relative to the DNA base sequence, and the number of resulting strand breaks in each nucleotide is determined. As the intensity of damage declines consistently with distance from the radionuclide, relative distances between the emitter and the nucleotides can be deduced, and hence potentially the topology or structural detail of the DNA. For simulation, appropriate software includes a Molecular Dynamics package, analysis and visualization tools, and a Monte Carlo track structure program. CONCLUSIONS: A review of published work and our own recent unpublished studies have shown that radioprobing is sufficiently sensitive and consistent to determine structural detail such as internal folding topology and flexing behavior, and can be applied to DNA or a DNA-protein complex in an approximation to its normal biological environment.

Simulation of (125)I-based radioprobing experiments to study DNA quadruplex structure and topology.

PURPOSE: Certain guanine-rich DNA sequences have the capacity to fold into four-stranded structures stabilized by the stacking of square planar arrangements of four hydrogen-bonded guanine bases. However both the overall topology of folding and the more detailed three dimensional structure of these quadruplexes is difficult to determine or predict, and they can be polymorphic, altering radically depending on environmental conditions. Radioprobing experiments, in which Auger electrons emitted during the decay of a (125)I-containing base induce strand cleavage in a distance- and structure-dependent manner, have provided possible means of determining these details. Here we have used a combination of computer simulation methods to study the information obtained by one such experiment, reported in 2004. METHOD: Models were constructed of three quadruplex topologies considered in the experiment, and one other topology proposed more recently. Molecular Dynamics simulations were used to equilibrate these structures and monitor how they evolved over several nanoseconds in solution. Snapshots from the trajectories were then subjected to Monte Carlo track structure prediction, from which theoretical cleavage patterns have been extracted. RESULTS: The four topologies were found to yield quite different cleavage patterns, which allow the presence of particular conformations in an experiment to be predicted. CONCLUSION: Radioprobing, which is usable in biologically relevant environments, is sensitive enough to distinguish with some confidence between alternative folding topologies in a DNA structure. Monte Carlo track structure simulation can reinforce or question conclusions drawn from experiment, and Molecular Dynamics used with various restraints provides a practical means of guiding a model towards one that yields cleavage patterns closer to those found experimentally.