Spatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break

Abstract

Tumor suppressor p53-binding protein 1 (53BP1) is a DNA repair protein essential for the detection, assessment, and resolution of DNA double strand breaks (DSBs). The presence of a DSB is signaled to 53BP1 via a local histone modification cascade that triggers the binding of 53BP1 dimers to chromatin flanking this type of lesion. While biochemical studies have established that 53BP1 exists as a dimer, it has never been shown in a living cell when or where 53BP1 dimerizes upon recruitment to a DSB site, or upon arrival at this nuclear location, how the DSB histone code to which 53BP1 dimers bind regulates retention and self-association into higher-order oligomers. Thus, here in live-cell nuclear architecture we quantify the spatiotemporal dynamics of 53BP1 oligomerization during a DSB DNA damage response by coupling fluorescence fluctuation spectroscopy (FFS) with the DSB inducible via AsiSI cell system (DIvA). From adopting this multiplexed approach, we find that preformed 53BP1 dimers relocate from the nucleoplasm to DSB sites, where consecutive recognition of ubiquitinated lysine 15 of histone 2A (H2AK15ub) and di-methylated lysine 20 of histone 4 (H4K20me2), leads to the assembly of 53BP1 oligomers and a mature 53BP1 foci structure.

Topoisomerase I inhibition and peripheral nerve injury induce DNA breaks and ATF3-associated axon regeneration in sensory neurons

“…Similar DNA break foci (as detected by 53BP1 and g-H2AX overlapping puncta), albeit fewer per neuron, were also found in the nuclei of DRG neurons as early as 10 min after a sciatic nerve crush injury (Figure 5A). Although the 53BP1 staining appears fuzzy in the nucleus, it localizes to foci in injured DRG neurons, which is the same as seen in other cell types (Harrigan et al, 2011;Lou et al, 2020;Shanbhag et al, 2019). The peak percentage of neurons with DSD foci occurred 30 min after the nerve injury, with a second but smaller increase at 24 h after the injury (Figure 5B).…”

Live cell dynamics of the NF-Y transcription factor

“…To determine the stoichiometry of the NF-Y complex that was indirectly measured by consecutive FLIM-FRET detection of YA/YB/YC interaction in live cells, we next transfected HeLa cells with eGFP-YA, eGFP-YB or eGFP-YC and measured their respective oligomeric states via Number and Brightness (NB) analysis (Fig. 2 A–C) 30 , 31 . From comparison of the apparent brightness of eGFP-YB, eGFP-YC and eGFP-YA with the apparent brightness of our monomeric control eGFP we find each NF-Y subunit to be monomeric (Fig.…”

VRK1 Depletion Facilitates the Synthetic Lethality of Temozolomide and Olaparib in Glioblastoma Cells

“…Accumulation and 53BP1 Recruitment to Damage Sites Induced by TMZ and Olaparib NHEJ is the main repair pathway of DSB (Pannunzio et al, 2018;Zhao et al, 2020). An intermediate step in the NHEJ pathway, is mediated by the formation of 53BP1 foci at DNA damage sites (Sanz-Garcia et al, 2012), which requires the accumulation of H4K20me2 (Jacquet et al, 2016;Guo et al, 2018;Lou et al, 2020). Therefore, we studied the effect of VRK1 depletion on the accumulation of H4K20me2, and the formation 53BP1 foci induced by TMZ and olaparib.…”