Pryde Fiona, Khalili Shirin, Robertson Kathryn, Selfridge Jim, Ritchie Ann-Marie, Melton David W, Jullien Denis, Adachi Yasuhisa
The Wellcome Trust Centre for Cell Biology, The Institute of Cell and Molecular Biology, The University of Edinburgh, The King's Buildings, Edinburgh, EH9 3JR, UK.
J Cell Sci. 2005 May 1;118(Pt 9):2043-55. doi: 10.1242/jcs.02336. Epub 2005 Apr 19.
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.
53BP1蛋白在电离辐射（IR）后会重新定位于DNA损伤位点，并参与DNA损伤检查点信号转导。我们检测了活细胞中GFP-53BP1的动态变化。损伤诱导后2-3分钟，该蛋白开始在DNA损伤位点积累。光漂白实验后的荧光恢复表明，GFP-53BP1在未受辐射的细胞中具有高度的流动性。与IR诱导的核灶结合后，53BP1的流动性大大降低。53BP1靶向IR诱导灶所必需的最小（M）结构域由1220-1703位氨基酸残基组成。GFP-M蛋白在缺乏功能性内源性53BP1的小鼠胚胎成纤维细胞中形成灶。M结构域包含一个Tudor基序的串联重复序列和一个富含精氨酸和甘氨酸的结构域（RG序列），这些在参与RNA代谢的蛋白质中经常出现，前者对于靶向是必需的。RNase A处理可使53BP1从IR诱导的灶中解离。在HeLa细胞中，RNase A处理导致的不含RG序列的M结构域的解离在辐射后早期通过重新添加核RNA可以恢复。53BP1免疫沉淀复合物中含有一些RNA分子。我们的结果表明RNA可能参与了53BP1与IR损伤染色质的结合。
