53BP1 exchanges slowly at the sites of DNA damage and appears to require RNA for its association with chromatin
53BP1 exchanges slowly at the sites of DNA damage and appears to require RNA for its association with chromatin
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DOI：
10.1242/jcs.02336
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发表时间：
2005-05-01
影响因子：
4
通讯作者：
Adachi, Y
中科院分区：
文献类型：
--
作者：
Pryde, F;Khalili, S;Adachi, Y
53BP1蛋白在电离辐射（IR）后重新定位到DNA损伤位点，并参与DNA损伤检查点信号转导。我们检测了GFP-53BP1在活细胞中的动态。损伤诱导后2-3分钟，蛋白质开始在DNA损伤部位聚集。光漂白实验后的荧光恢复表明，GFP-53BP1在未照射的细胞中具有很高的流动性。53BP1与红外诱导的核病灶结合后，其迁移率大大降低。53BP1的最小(M)结构域由残基1220-1703组成，是靶向红外诱导病灶所必需的。GFP-M蛋白在缺乏内源性53BP1的小鼠胚胎成纤维细胞中形成病灶。M结构域包含Tudor基序的串联重复序列和富含精氨酸和甘氨酸的结构域（RG stretch），它们通常存在于参与RNA代谢的蛋白质中，前者对于靶向至关重要。RNase A治疗可将53BP1从ir诱导的病灶中分离出来。在HeLa细胞中，经过RNase A处理的无RG拉伸的M结构域解离可以在辐照后的早期阶段通过重新添加核RNA来恢复。53BP1免疫沉淀物含有一些RNA分子。我们的研究结果表明，RNA可能参与了53BP1与IR损伤的染色质的结合。
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.