DNA repair in the context of chromatin: new molecular insights by the nanoscale detection of DNA repair complexes using transmission electron microscopy
| Autor: | Nadine Schuler, Stefanie Schanz, Claudia E. Rübe, Gunther Wennemuth, Christian Rübe, Yvonne Lorat |
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| Rok vydání: | 2010 |
| Předmět: |
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DNA Repair DNA repair Chromosomal Proteins Non-Histone Metal Nanoparticles Biochemistry Homology directed repair Mice Microscopy Electron Transmission Animals DNA Breaks Double-Stranded Molecular Biology Replication protein A chemistry.chemical_classification Cell Nucleus Neurons DNA ligase biology Nuclear Proteins Cell Biology Molecular biology Chromatin Cell biology DNA-Binding Proteins Mice Inbred C57BL Kinetics Histone chemistry biology.protein DNA mismatch repair Tumor Suppressor p53-Binding Protein 1 Whole-Body Irradiation |
| Zdroj: | DNA repair. 10(4) |
| ISSN: | 1568-7856 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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