Abstract
The spatial distribution of DSB repair factors γH2AX, 53BP1 and Rad51 in ionizing radiation induced foci (IRIF) in HeLa cells using super resolution STED nanoscopy after low and high linear energy transfer (LET) irradiation was investigated. 53BP1 and γH2AX form IRIF with same mean size of (540 ± 40) nm after high LET irradiation while the size after low LET irradiation is significantly smaller. The IRIF of both repair factors show nanostructures with partial anti-correlation. These structures are related to domains formed within the chromatin territories marked by γH2AX while 53BP1 is mainly situated in the perichromatin region. The nanostructures have a mean size of (129 ± 6) nm and are found to be irrespective of the applied LET and the labelled damage marker. In contrast, Rad51 shows no nanostructure and a mean size of (143 ± 13) nm independent of LET. Although Rad51 is surrounded by 53BP1 it strongly anti-correlates meaning an exclusion of 53BP1 next to DSB when decision for homologous DSB repair happened.
Highlights
Repetition 2 2 2 1 filamentous structures and links the complementary chromatin strands together[5,6]
The low linear energy transfer (LET) experiment for 53BP1-γH 2AX was performed in one repetition with 20 ionizing radiation induced foci (IRIF), which is reasonable to compare to the other results
The IRIF from proton irradiation are spread over the whole cell nucleus as expected from a random low LET radiation where the induced double-strand breaks (DSB) are produced independently from one another
Summary
Repetition 2 2 2 1 filamentous structures and links the complementary chromatin strands together[5,6]. Ochs and co-workers conclude the functional connection through experiments using a knock-down of 53BP1, which resulted in a distinct lack of Rad[51] promoted repair and was further verified through colocalization analysis of these two proteins in confocal microscopy This colocalization at IRIF turned out to be a nanoscopic anticorrelation in the detailed structures, when imaged with better resolution[13]. This border zone is functionally important, as first evidence suggests that it is the location where amongst other processes, transcription, DNA replication and DNA repair occur[25] This region has a size of 100–200 nm[24] and is not totally filled with chromatin but offers room for proteins binding to DNA. Using the improved resolution of the applied STED nanoscopy as compared to the previous study[13] and utilizing quantitative size and correlation analysis a detailed insight into the structural and functional relationships of DSB repair factors occurring on the nanoscale was obtained
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