DNA repair in the context of chromatin: new molecular insights by the nanoscale detection of DNA repair complexes using transmission electron microscopy.

The recognition and repair of DNA double-strand breaks (DSBs) occurs in the context of highly structured chromatin. Here, we established a transmission electron microscopy (TEM) approach to localize gold-labeled DSB repair components in different chromatin environments within the intact nuclear architecture of cells in irradiated mouse tissues. The ultra-high resolution of TEM offers the intriguing possibility of detecting core components of the DNA repair machinery at the single-molecule level and visualizing their molecular interactions with specific histone modifications. By labeling phosphorylated Ku70, which binds directly to broken DNA ends in preparation for rejoining, this TEM approach can monitor formation and repair of actual DSBs in euchromatic versus heterochromatic regions. While DNA lesions in euchromatin are detected and rejoined without any delay, DNA packaging in heterochromatin appears to retard DSB processing, leading to slower repair kinetics. Of significance, the assembly of γH2AX, MDC1, and 53BP1 occurs exclusively at DSBs in heterochromatic (characterized by H3K9me3), but not euchromatic domains, suggesting involvement in localized chromatin decondensation (which increases heterochromatic DNA accessibility). Collectively, this TEM approach provides fascinating insights into the dynamic events of the DSB repair process that depend decisively upon the actual chromatin structure around the break.

[1]  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.

[2]  G. Taucher‐Scholz,et al.  Positional Stability of Damaged Chromatin Domains along Radiation Tracks in Mammalian Cells , 2009, Radiation research.

[3]  C. Dinant,et al.  The Emerging Role of HP1 in the DNA Damage Response , 2009, Molecular and Cellular Biology.

[4]  M. Nussenzweig,et al.  Chromatin dynamics and the preservation of genetic information , 2007, Nature.

[5]  M. Durante,et al.  Live cell microscopy analysis of radiation-induced DNA double-strand break motion , 2009, Proceedings of the National Academy of Sciences.

[6]  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.

[7]  Stephen J. Elledge,et al.  MDC1 is a mediator of the mammalian DNA damage checkpoint , 2003, Nature.

[8]  Markus Löbrich,et al.  γH2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization , 2010, Cell cycle.

[9]  J. Dahm-Daphi,et al.  Distinct roles of XRCC4 and Ku80 in non-homologous end-joining of endonuclease- and ionizing radiation-induced DNA double-strand breaks , 2008, Nucleic acids research.

[10]  A. Nussenzweig,et al.  ATM breaks into heterochromatin. , 2008, Molecular cell.

[11]  G. Almouzni,et al.  HP1 and the dynamics of heterochromatin maintenance , 2004, Nature Reviews Molecular Cell Biology.

[12]  C. Rübe,et al.  DNA double-strand break rejoining in complex normal tissues. , 2008, International journal of radiation oncology, biology, physics.

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

[14]  J. Wang,et al.  DNA looping by Ku and the DNA-dependent protein kinase. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Ashby J. Morrison,et al.  DNA Repair in the Context of Chromatin , 2005, Cell cycle.

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

[17]  J. Walker,et al.  Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair , 2001, Nature.

[18]  T. Misteli,et al.  The emerging role of nuclear architecture in DNA repair and genome maintenance , 2009, Nature Reviews Molecular Cell Biology.

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

[20]  R. Schneider,et al.  Dynamics and interplay of nuclear architecture, genome organization, and gene expression. , 2007, Genes & development.

[21]  K M Prise,et al.  A review of dsb induction data for varying quality radiations. , 1998, International journal of radiation biology.

[22]  Yves Pommier,et al.  The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks , 2010, Cell cycle.

[23]  A. Shibata,et al.  53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair , 2010, Nature Cell Biology.

[24]  P. Jeggo,et al.  The impact of heterochromatin on DSB repair. , 2009, Biochemical Society transactions.

[25]  O. Fernandez-Capetillo,et al.  Why cells respond differently to DNA damage: A chromatin perspective , 2008, Cell cycle.

[26]  J. Dahm-Daphi,et al.  The alternative end-joining pathway for repair of DNA double-strand breaks requires PARP1 but is not dependent upon microhomologies , 2010, Nucleic acids research.

[27]  G. Iliakis,et al.  Mechanisms of DNA double strand break repair and chromosome aberration formation , 2004, Cytogenetic and Genome Research.

[28]  C. Rübe,et al.  Accumulation of DNA double-strand breaks in normal tissues after fractionated irradiation. , 2010, International journal of radiation oncology, biology, physics.

[29]  L. Argaman,et al.  The Direct Interaction between 53BP1 and MDC1 Is Required for the Recruitment of 53BP1 to Sites of Damage* , 2009, Journal of Biological Chemistry.

[30]  J. Bartek,et al.  The DNA-damage response in human biology and disease , 2009, Nature.

[31]  B. Stenerlöw,et al.  Rejoining of double-stranded DNA-fragments studied in different size-intervals , 2002, International journal of radiation biology.

[32]  S. Lees-Miller,et al.  Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining. , 2009, The Biochemical journal.

[33]  M. Barcellos-Hoff,et al.  Spatiotemporal characterization of ionizing radiation induced DNA damage foci and their relation to chromatin organization. , 2010, Mutation research.

[34]  T. Kouzarides.  Chromatin Modifications and Their Function , 2007, Cell.

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

[36]  R. Bristow,et al.  Microscopic imaging of DNA repair foci in irradiated normal tissues , 2009, International journal of radiation biology.

[37]  Seung-Min Yang,et al.  Global chromatin compaction limits the strength of the DNA damage response , 2007, The Journal of cell biology.

[38]  Qianzheng Zhu,et al.  Histone modifications: crucial elements for damage response and chromatin restoration , 2010, Journal of cellular physiology.

[39]  T. Misteli.  Beyond the Sequence: Cellular Organization of Genome Function , 2011 .

[40]  M. Löbrich,et al.  DNA Double-Strand Break Repair of Blood Lymphocytes and Normal Tissues Analysed in a Preclinical Mouse Model: Implications for Radiosensitivity Testing , 2008, Clinical Cancer Research.

[41]  S. Gasser,et al.  Crosstalk between histone modifications during the DNA damage response. , 2009, Trends in cell biology.