Image-Based Modeling Reveals Dynamic Redistribution of DNA Damage into Nuclear Sub-Domains

Several proteins involved in the response to DNA double strand breaks (DSB) form microscopically visible nuclear domains, or foci, after exposure to ionizing radiation. Radiation-induced foci (RIF) are believed to be located where DNA damage occurs. To test this assumption, we analyzed the spatial distribution of 53BP1, phosphorylated ATM, and γH2AX RIF in cells irradiated with high linear energy transfer (LET) radiation and low LET. Since energy is randomly deposited along high-LET particle paths, RIF along these paths should also be randomly distributed. The probability to induce DSB can be derived from DNA fragment data measured experimentally by pulsed-field gel electrophoresis. We used this probability in Monte Carlo simulations to predict DSB locations in synthetic nuclei geometrically described by a complete set of human chromosomes, taking into account microscope optics from real experiments. As expected, simulations produced DNA-weighted random (Poisson) distributions. In contrast, the distributions of RIF obtained as early as 5 min after exposure to high LET (1 GeV/amu Fe) were non-random. This deviation from the expected DNA-weighted random pattern can be further characterized by “relative DNA image measurements.” This novel imaging approach shows that RIF were located preferentially at the interface between high and low DNA density regions, and were more frequent than predicted in regions with lower DNA density. The same preferential nuclear location was also measured for RIF induced by 1 Gy of low-LET radiation. This deviation from random behavior was evident only 5 min after irradiation for phosphorylated ATM RIF, while γH2AX and 53BP1 RIF showed pronounced deviations up to 30 min after exposure. These data suggest that DNA damage–induced foci are restricted to certain regions of the nucleus of human epithelial cells. It is possible that DNA lesions are collected in these nuclear sub-domains for more efficient repair.

[1]  Bo Stenerlöw,et al.  Measurement of Prompt DNA Double-Strand Breaks in Mammalian Cells without Including Heat-Labile Sites: Results for Cells Deficient in Nonhomologous End Joining , 2002, Radiation research.

[2]  T. Halazonetis,et al.  P53 Binding Protein 1 (53bp1) Is an Early Participant in the Cellular Response to DNA Double-Strand Breaks , 2000, The Journal of cell biology.

[3]  Michael J. Hendzel,et al.  Quantitative Analysis Reveals Asynchronous and more than DSB-Associated Histone H2AX Phosphorylation after Exposure to Ionizing Radiation , 2006, Radiation research.

[4]  R Eils,et al.  Compartmentalization of interphase chromosomes observed in simulation and experiment. , 1999, Journal of molecular biology.

[5]  George Iliakis,et al.  Computational Methods for Analysis of Foci: Validation for Radiation-Induced γ-H2AX Foci in Human Cells , 2006, Radiation research.

[6]  P. Olive,et al.  Hypertonic Saline Enhances Expression of Phosphorylated Histone H2AX after Irradiation , 2004, Radiation research.

[7]  T. Cremer,et al.  Chromosome territories, nuclear architecture and gene regulation in mammalian cells , 2001, Nature Reviews Genetics.

[8]  James G. McNally,et al.  Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks , 2006, The Journal of cell biology.

[9]  Y. Adachi,et al.  Phosphorylation and Rapid Relocalization of 53BP1 to Nuclear Foci upon DNA Damage , 2001, Molecular and Cellular Biology.

[10]  Junjie Chen,et al.  Accumulation of Checkpoint Protein 53BP1 at DNA Breaks Involves Its Binding to Phosphorylated Histone H2AX* , 2003, Journal of Biological Chemistry.

[11]  R K Sachs,et al.  Locations of radiation-produced DNA double strand breaks along chromosomes: a stochastic cluster process formalism. , 1999, Mathematical biosciences.

[12]  Adayabalam S Balajee,et al.  Replication protein A and gamma-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks. , 2004, Experimental cell research.

[13]  Anne E Carpenter,et al.  Long-Range Directional Movement of an Interphase Chromosome Site , 2006, Current Biology.

[14]  Francis A. Cucinotta,et al.  Novel image processing interface to relate DSB spatial distribution from experiments with phosphorylation foci to the state-of-the-art models of DNA breakage , 2006 .

[15]  Jean Gautier,et al.  Two-step activation of ATM by DNA and the Mre11–Rad50–Nbs1 complex , 2006, Nature Structural &Molecular Biology.

[16]  Junjie Chen,et al.  Tumor Suppressor P53 Binding Protein 1 (53bp1) Is Involved in DNA Damage–Signaling Pathways , 2001, The Journal of cell biology.

[17]  J R Savage,et al.  Insight into sites. , 1996, Mutation research.

[18]  M Scholz,et al.  Biological Imaging of Heavy Charged-Particle Tracks , 2003, Radiation research.

[19]  Francis A Cucinotta,et al.  Chromatin loops are responsible for higher counts of small DNA fragments induced by high-LET radiation, while chromosomal domains do not affect the fragment sizes , 2006, International journal of radiation biology.

[20]  Martha R. Stampfer,et al.  Isolation and growth of human mammary epithelial cells , 1985 .

[21]  S. Lees-Miller,et al.  DNA damage-induced activation of ATM and ATM-dependent signaling pathways. , 2004, DNA repair.

[22]  Michel C. Nussenzweig,et al.  Genomic Instability in Mice Lacking Histone H2AX , 2002, Science.

[23]  Ying-li Yu,et al.  Cell Cycle-Dependent Expression of Phosphorylated Histone H2AX: Reduced Expression in Unirradiated but not X-Irradiated G1-Phase Cells , 2003, Radiation research.

[24]  Y. Shiloh,et al.  ATM: sounding the double-strand break alarm. , 2000, Cold Spring Harbor symposia on quantitative biology.

[25]  Jiri Bartek,et al.  Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks , 2006, The Journal of cell biology.

[26]  M. Kastan,et al.  DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation , 2003, Nature.

[27]  E. Rogakou,et al.  DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139* , 1998, The Journal of Biological Chemistry.

[28]  R K Sachs,et al.  A polymer, random walk model for the size-distribution of large DNA fragments after high linear energy transfer radiation , 2000, Radiation and environmental biophysics.

[29]  V. Yamazaki,et al.  A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage , 2000, Current Biology.

[30]  Leif E. Peterson,et al.  Extrapolation of the DNA Fragment-Size Distribution after High-Dose Irradiation to Predict Effects at Low Doses , 2001, Radiation research.

[31]  F A Cucinotta,et al.  Applications of amorphous track models in radiation biology , 1999, Radiation and environmental biophysics.

[32]  Y. Shiloh,et al.  Nuclear retention of ATM at sites of DNA double strand breaks. , 2001, The Journal of biological chemistry.

[33]  J. Savage,et al.  Reflections and meditations upon complex chromosomal exchanges. , 2002, Mutation research.

[34]  W. J. Meath,et al.  Dipole spectrum of water vapor and its relation to the energy loss of fast-charged particles. , 1975, Radiation research.

[35]  J. L. Magee,et al.  RADIATION CHEMISTRY OF HEAVY PARTICLE TRACKS. I. GENERAL CONSIDERATIONS , 1980 .

[36]  F A Cucinotta,et al.  Model for Radial Dependence of Frequency Distributions for Energy Imparted in Nanometer Volumes from HZE Particles , 2000, Radiation research.

[37]  T Hyslop,et al.  DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus. , 2000, Cancer research.

[38]  P. Olive,et al.  Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X‐rays , 2003, International journal of radiation biology.

[39]  M. Hendzel,et al.  H1 Family Histones in the Nucleus , 2005, Journal of Biological Chemistry.

[40]  Bo Stenerlöw,et al.  Focus Formation of DNA Repair Proteins in Normal and Repair-Deficient Cells Irradiated with High-LET Ions , 2004, Radiation research.

[41]  Rodney Rothstein,et al.  Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre , 2003, Nature Cell Biology.

[42]  Sylvain V Costes,et al.  Automatic and quantitative measurement of protein-protein colocalization in live cells. , 2004, Biophysical journal.

[43]  Bahram Parvin,et al.  Imaging Features that Discriminate between Foci Induced by High- and Low-LET Radiation in Human Fibroblasts , 2006, Radiation research.

[44]  J. Aten,et al.  Dynamics of DNA Double-Strand Breaks Revealed by Clustering of Damaged Chromosome Domains , 2004, Science.