Abstract
PurposeDNA double-strand breaks (DSBs) generated by ionizing radiation pose a serious threat to the preservation of genetic and epigenetic information. The known importance of local chromatin configuration in DSB repair raises the question of whether breaks in different chromatin environments are recognized and repaired by the same repair machinery and with similar efficiency. An essential step in DSB processing by non-homologous end joining is the high-affinity binding of Ku70-Ku80 and DNA-PKcs to double-stranded DNA ends that holds the ends in physical proximity for subsequent repair.Methods and MaterialsUsing transmission electron microscopy to localize gold-labeled pKu70 and pDNA-PKcs within nuclear ultrastructure, we monitored the formation and repair of actual DSBs within euchromatin (electron-lucent) and heterochromatin (electron-dense) in cortical neurons of irradiated mouse brain.ResultsWhile DNA lesions in euchromatin (characterized by two pKu70-gold beads, reflecting the Ku70-Ku80 heterodimer) are promptly sensed and rejoined, DNA packaging in heterochromatin appears to retard DSB processing, due to the time needed to unravel higher-order chromatin structures. Complex pKu70-clusters formed in heterochromatin (consisting of 4 or ≥6 gold beads) may represent multiple breaks in close proximity caused by ionizing radiation of highly-compacted DNA. All pKu70-clusters disappeared within 72 hours post-irradiation, indicating efficient DSB rejoining. However, persistent 53BP1 clusters in heterochromatin (comprising ≥10 gold beads), occasionally co-localizing with γH2AX, but not pKu70 or pDNA-PKcs, may reflect incomplete or incorrect restoration of chromatin structure rather than persistently unrepaired DNA damage.DiscussionHigher-order organization of chromatin determines the accessibility of DNA lesions to repair complexes, defining how readily DSBs are detected and processed. DNA lesions in heterochromatin appear to be more complex, with multiple breaks in spatial vicinity inducing severe chromatin disruptions. Imperfect restoration of chromatin configurations may leave DSB-induced epigenetic memory of damage with potentially pathological repercussions.
Highlights
DNA double-strand breaks (DSBs) generated by ionizing radiation represent an extremely cytolethal form of DNA damage and pose a serious threat to the preservation of genetic and epigenetic information
Complex phosphorylated Ku70 (pKu70)-clusters formed in heterochromatin may represent multiple breaks in close proximity caused by ionizing radiation of highly-compacted DNA
Higher-order organization of chromatin determines the accessibility of DNA lesions to repair complexes, defining how readily DSBs are detected and processed
Summary
DNA double-strand breaks (DSBs) generated by ionizing radiation represent an extremely cytolethal form of DNA damage and pose a serious threat to the preservation of genetic and epigenetic information. Cells have evolved complex DNA damage response (DDR) mechanisms to ensure genomic integrity that use signaling networks to sense DSBs, arrest the cell cycle, activate DNA repair processes, and, restore the original chromatin structure. DNA-bound Ku directs the recruitment of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) via a small helical domain at the C terminus of Ku80, resulting in the assembly of the holoenzyme DNA-PK and activation of its kinase activity. This DNA-PK complex keeps broken DNA ends in close proximity and proper alignment, providing a recruitment platform for subsequent repair factors [1]
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