53BP1 exchanges slowly at the sites of DNA damage and appears to require RNA for its association with chromatin

Abstract

53BP1 protein is re-localized to the sites of DNA damage after ionizing radiation (IR) and is involved in DNA-damage-checkpoint signal transduction. We examined the dynamics of GFP-53BP1 in living cells. The protein starts to accumulate at the sites of DNA damage 2-3 minutes after damage induction. Fluorescence recovery after photobleaching experiments showed that GFP-53BP1 is highly mobile in non-irradiated cells. Upon binding to the IR-induced nuclear foci, the mobility of 53BP1 reduces greatly. The minimum (M) domain of 53BP1 essential for targeting to IR induced foci consists of residues 1220-1703. GFP-M protein forms foci in mouse embryonic fibroblast cells lacking functional endogenous 53BP1. The M domain contains a tandem repeat of Tudor motifs and an arginine- and glycine-rich domain (RG stretch), which are often found in proteins involved in RNA metabolism, the former being essential for targeting. RNase A treatment dissociates 53BP1 from IR-induced foci. In HeLa cells, dissociation of the M domain without the RG stretch by RNase A treatment can be restored by re-addition of nuclear RNA in the early stages of post-irradiation. 53BP1 immunoprecipitates contain some RNA molecules. Our results suggest a possible involvement of RNA in the binding of 53BP1 to chromatin damaged by IR.

ATR and Rad17 collaborate in modulating Rad9 localisation at sites of DNA damage

“…Therefore, we assessed whether Rad9 proteins within established foci are immobile or whether these foci are more dynamic structures in which Rad9 proteins turnover, a process that could possibly be affected by ATR. To assess this, we used simultaneous fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP) technology, an assay used in living cells that has been successfully used to monitor protein redistribution in real time (Essers et al, 2005;Hoogstraten et al, 2002;Lukas et al, 2003;Mattern et al, 2004;Pryde et al, 2005). In these experiments, U2OS cells expressing GFP-Rad9 were transfected with control or ATR siRNA oligonucleotides.…”

RNF8-dependent and RNF8-independent Regulation of 53BP1 in Response to DNA Damage

“…4A) (35). This fragment of 53BP1 spans the Tudor domain, which targets 53BP1 to foci through interaction with dimethylated histone H4 (11).…”

A Tandem of SH3-like Domains Participates in RNA Binding in KIN17, a Human Protein Activated in Response to Genotoxics

“…Furthermore, the association of a region of 53BP1, containing the tandem Tudor domains, with IR-induced foci is sensitive to treatment with ribonuclease, suggesting that another function of double Tudor of 53BP1 is to bind RNA. 48 Thus, RNA binding could represent a more general function of tandem SH3-like domains. In this study, a novel RNA-binding site in a double SH3-like domain is characterized even if it does not exclude the possibility that this domain is involved also in protein-protein interaction.…”