γH2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization

DNA double-strand breaks (DSBs) represent an important radiation-induced lesion and impaired DSB repair provides the best available correlation with radiosensitivity. Physical techniques for monitoring DSB repair require high, non-physiological doses and cannot reliably detect subtle defects. One outcome from extensive research into the DNA damage response is the observation that H2AX, a variant form of the histone H2A, undergoes extensive phosphorylation at the DSB, creating γH2AX foci that can be visualised by immunofluorescence. There is a close correlation between γH2AX foci and DSB numbers and between the rate of foci loss and DSB repair, providing a sensitive assay to monitor DSB repair in individual cells using physiological doses. However, γH2AX formation can occur at single-stranded DNA regions which arise during replication or repair and thus does not solely correlate with DSB formation. Here, we present and discuss evidence that following exposure to ionising radiation, γH2AX foci analysis can provide a sensitive monitor of DSB formation and repair and describe techniques to optimise the analysis. We discuss the limitations and benefits of the technique, enabling the procedure to be optimally exploited but not misused.

[1]  Stephen J. Elledge,et al.  Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes , 2003, Science.

[2]  Kai Rothkamm,et al.  Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[3]  A. Gudkov,et al.  Pseudo-DNA damage response in senescent cells , 2009, Cell cycle.

[4]  P. Jeggo,et al.  ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin. , 2008, Molecular cell.

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

[6]  Michael Uder,et al.  In vivo formation and repair of DNA double-strand breaks after computed tomography examinations. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[7]  P. Jeggo,et al.  An imperfect G 2 M checkpoint contributes to chromosome instability following irradiation of S and G 2 phase cells , 2007 .

[8]  P. Jeggo,et al.  The impact of a negligent G2/M checkpoint on genomic instability and cancer induction , 2007, Nature Reviews Cancer.

[9]  D. Durocher,et al.  Regulatory ubiquitylation in response to DNA double-strand breaks. , 2009, DNA repair.

[10]  Yair Andegeko,et al.  Requirement of the MRN complex for ATM activation by DNA damage , 2003, The EMBO journal.

[11]  Stephen P. Jackson,et al.  Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage , 2005, Nature.

[12]  P. Jeggo,et al.  Chromosome breakage after G2 checkpoint release , 2007, The Journal of cell biology.

[13]  L. Zou,et al.  Single-stranded DNA orchestrates an ATM-to-ATR switch at DNA breaks. , 2009, Molecular cell.

[14]  W. Bonner,et al.  γ-H2AX in Cancer Cells: A Potential Biomarker for Cancer Diagnostics, Prediction and Recurrence , 2006, Cell cycle.

[15]  P. Jeggo,et al.  X-irradiation of cells on glass slides has a dose doubling impact. , 2007, DNA repair.

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

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

[18]  G. Iliakis,et al.  Measurement of DNA double-strand breaks in CHO cells at various stages of the cell cycle using pulsed field gel electrophoresis: calibration by means of 125I decay. , 1991, International journal of radiation biology.

[19]  T. Lange,et al.  Cell Cycle-Dependent Role of MRN at Dysfunctional Telomeres: ATM Signaling-Dependent Induction of Nonhomologous End Joining (NHEJ) in G 1 and Resection-Mediated Inhibition of NHEJ in G 2 † , 2009 .

[20]  Markus Löbrich,et al.  ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2 , 2009, The EMBO journal.

[21]  J. Dahm-Daphi,et al.  Correlation between cellular radiosensitivity and non-repaired double-strand breaks studied in nine mammalian cell lines. , 2009, International journal of radiation biology.

[22]  M. Durante,et al.  Chromosome condensation outside of mitosis: Mechanisms and new tools , 2006, Journal of cellular physiology.

[23]  Martin Kühne,et al.  A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci. , 2004, Molecular cell.

[24]  A. Carr,et al.  Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism. , 2009, Genes & development.

[25]  P. Russell,et al.  Mre11 Dimers Coordinate DNA End Bridging and Nuclease Processing in Double-Strand-Break Repair , 2008, Cell.

[26]  Kai Rothkamm,et al.  A Double-Strand Break Repair Defect in ATM-Deficient Cells Contributes to Radiosensitivity , 2004, Cancer Research.

[27]  Kai Rothkamm,et al.  Pathways of DNA Double-Strand Break Repair during the Mammalian Cell Cycle , 2003, Molecular and Cellular Biology.

[28]  Jeroen A. A. Demmers,et al.  Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4 , 2006, Proceedings of the National Academy of Sciences.

[29]  K. Kohn,et al.  X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution. , 1979, Nucleic acids research.

[30]  Aaron Ponti,et al.  Replication foci dynamics: replication patterns are modulated by S‐phase checkpoint kinases in fission yeast , 2007, The EMBO journal.

[31]  B. Rydberg.  Radiation-Induced Heat-Labile Sites that Convert into DNA Double-Strand Breaks , 2000, Radiation research.

[32]  George Iliakis,et al.  γ-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin , 2008, Nucleic acids research.

[33]  R. Kanaar,et al.  DNA double-strand break repair: all's well that ends well. , 2006, Annual review of genetics.

[34]  S. Jackson,et al.  Activation of the DNA Damage Response by Telomere Attrition: A Passage to Cellular Senescence , 2004, Cell cycle.

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

[36]  John A. Tainer,et al.  Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair , 2009, Cell.

[37]  Jiri Bartek,et al.  ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks , 2006, Nature Cell Biology.