Chromatin organization revealed by nanostructure of irradiation induced γH2AX, 53BP1 and Rad51 foci

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.Ionizing radiation induces a variety of different types of damage when targeted to living cells. Severe damage, which can influence cell survival or lead to carcinogenesis, occurs due to ionizing events in the DNA molecule itself. The most lethal of these types of DNA damages are the double-strand breaks (DSB), as they may lead to genetic alterations which in turn can be responsible for cell death or carcigonesis. Mammalian cells react with a variety of complex response mechanisms to DSB induction. One main reaction is the phosphorylation of the histone variant H2AX at serine 139 (S139) to obtain γ H2AX through kinases such as ATM, ATR and DNA-PK 1 . The γ H2AX domains occur in mega-base-pair (Mbp) large regions of the chromatin around DSB 2-6 and can be visualized as so-called ionizing radiation induced foci (IRIF) 7 . The recruitment and activation of proteins due to damage induction can later on lead to the repair of DSB. The cell has different repair mechanisms to properly rejoin the ends of a DSB, including the possibly error-prone non-homologous end joining (NHEJ) 8 and the in most cases error-free homologous recombination (HR) 9 . HR is limited to the S/G2 cell cycle phase, due to the fact that a homologous sister chromatin is needed in close vicinity to the DSB as a template to repair the damaged chromatin 2-4 . As a backup pathway for failed NHEJ in G1 an alternative end-joining pathway (alt-EJ) has previously been identified, which works as a last resort, when the other pathways fail 8 . Recent work analyzed the clustering of DSB repair factors in detail using high resolution microscopy 10-12 and nanoscopy 11,[13][14][15][16] in combination with state of the art correlation and clustering analysis methods. With these methods it is possible to gain a deeper understanding of the functionality of DSB repair proteins and their interactions. After the first reactions to DSB induction, such as phosphorylation of H2AX (γ H2AX), the recruitment of downstream repair proteins starts for NHEJ as well as f...

Nanostructure of DNA repair foci revealed by superresolution microscopy

“…Despite numerous studies regarding the involvement of gH2AX in DNA repair, the nanoscale organization of gH2AX and DDR proteins within IR-induced foci remains largely unknown because of the resolution limit of conventional fluorescence microscopy. At present, there are only a few reported studies using superresolution fluorescence microscopy to explore the nanoscale organization of DSB repair foci (13)(14)(15)(16). Those studies revealed that DSB foci consist of several subfoci with a lateral diameter ranging between ;100 and 200 nm.…”

“…Because of its interactions with other NHEJ and non-NHEJ proteins, DNA-PKcs may also act as a scaffold protein to facilitate the localization of DNA-repair proteins to the site of DSBs (36). Unlike several other important DNA-repair proteins, such as, Ku70/80, 53BP1, Rad51, among others (12,14,16), DNA-PK has not yet been analyzed by superresolution microscopy.…”

Quantification of DNA damage induced γH2AX focus formation via super-resolution dSTORM localization microscopy

“…These steps allow the efficient recruitment of the repair factors to the damaged DNA regions and implicate in the choice between the DNA repair pathways. For examining the DSB-induced chromatin changes, confocal microscopy-based techniques are used in most of the studies, although in the last few years high-throughput chromosome conformation capture technique (4C) and single cell microscopy were utilized to gain detailed insights about the protein interactions and cascades involved in the different repair pathways 31,53–57 . A more detailed overview has raised more questions, which could be answered only at a single-cell level: how the different DNA repair pathways are chosen and how individual repair proteins are regulated to access the DNA repair site.…”

“…By using these techniques, it was shown that the γH2AX signal distributes up to a megabase around the damaged site 8 generating DNA repair foci with a typical feature size of half a micron, which is just above the resolution limit of traditional fluorescence microscopes. High resolution imaging based datasets of DSB structures have already been published and demonstrated via single molecule localization methods (SMLM) 23–28 , structured illumination microscopy (SIM) 26,29,30 , and stimulated emission depletion (STED) 29–31 super-resolution methods. These images can be further evaluated by using cluster analysis, and both the spatial distribution and the geometrical parameters of foci and even nano-foci can be determined 32,33 .…”

A change of view: homologous recombination at single-molecule resolution

“…It has been known for many years that repair foci demarcate the intracellular locations of recombination intermediates 142,148 , but we know very little about the precise organization and dynamic properties of the proteins, protein complexes and nucleic acid structures that form these foci 148 . A recent study using STED microscopy to study repair foci induced by ionizing radiation 149 demonstrates that SR imaging has the potential to provide a detailed picture of how the nucleoprotein complexes involved in recombination are organized in living cells.…”