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

Jieqiong Lou,Adèle Kerjouan,Ashleigh Solano,David G Priest,Elizabeth Hinde

doi:10.1038/s41467-020-19504-3

Jieqiong Lou, Adèle Kerjouan + Show 3 more

Open Access

https://doi.org/10.1038/s41467-020-19504-3

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Journal: Nature Communications	Publication Date: Nov 13, 2020
Citations: 26	License type: open-access

Affiliation: University of Melbourne

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.

Highlights

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)
From application of Number and Brightness (NB) and cross-pair correlation function (pCF) to DSB inducible via AsiSI cell system (DIvA) cells expressing different fluorescent constructs of 53BP1, and independent validation of this fluorescence fluctuation spectroscopy (FFS)-based strategy by fluorescence anisotropy imaging microscopy (FAIM) of homo-Förster resonance energy transfer (FRET)[39,40,41], we find the DSB histone code to regulate a spatiotemporal redistribution in 53BP1 oligomerization
In this study we coupled a multiplexed approach to FFS with the DIvA cell system, to investigate the spatiotemporal dynamics of 53BP1 dimer recruitment, retention, and oligomer formation at DSBs in a living cell

Summary

Introduction

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). 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. The biophysical mechanism by which 53BP1 molecules arrive at a chromatinsignaled DSB has not been directly observed within an intact cell nucleus, it is known from cryogenic electron microscopy that dimers of 53BP1 directly bind H2AK15ub-containing nucleosomes via the 53BP1 ubiquitylation-dependent recruitment motif and this interaction requires simultaneous engagement of the

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