Sp1 Facilitates DNA Double-Strand Break Repair through a Nontranscriptional Mechanism
Sp1 Facilitates DNA Double-Strand Break Repair through a Nontranscriptional Mechanism
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DOI：
10.1128/mcb.00049-12
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发表时间：
2012-09-01
影响因子：
5.3
通讯作者：
Azizkhan-Clifford, Jane
中科院分区：
文献类型：
--
作者：
Beishline, Kate;Kelly, Crystal M.;Azizkhan-Clifford, Jane
Sp1是一种广泛表达的转录因子，其在电离辐射和H2 O2的作用下被共济失调毛细血管扩张突变激酶（ATM）磷酸化。在这里，我们表明，通过间接免疫荧光，丝氨酸101（pSp 1）磷酸化的Sp1定位于电离辐射诱导的病灶与磷酸化组蛋白变体γ H2 Ax和成员的MRN（Mre 11，Rad 50，和Nbs 1）复合物。通过染色质免疫沉淀（ChIP）对DNA双链断裂（DSB）的占有率进行更精确的分析表明，Sp1与Nbs 1一样，位于DSB的200 bp范围内。利用激光微辐射的细胞，我们证明，pSp 1存在于DNA双链断裂诱导损伤后7.5分钟，并保持在断裂部位至少8小时。Sp1的耗尽抑制位点特异性DNA断裂的修复，并且N-末端182个氨基酸的肽（其含有ATM激酶的靶点但缺乏锌指DNA结合结构域）被磷酸化，定位于DSB，并且挽救由Sp1耗尽引起的修复缺陷。总之，这些数据表明，Sp1被迅速招募到紧邻DNA DSB位点的区域，并且是DSB修复所需的，通过一种独立于其序列指导的转录效应的机制。
Sp1 is a ubiquitously expressed transcription factor that is phosphorylated by ataxia telangiectasia mutated kinase (ATM) in response to ionizing radiation and H2O2. Here, we show by indirect immunofluorescence that Sp1 phosphorylated on serine 101 (pSp1) localizes to ionizing radiation-induced foci with phosphorylated histone variant gamma H2Ax and members of the MRN (Mre11, Rad50, and Nbs1) complex. More precise analysis of occupancy of DNA double-strand breaks (DSBs) by chromatin immunoprecipitation (ChIP) shows that Sp1, like Nbs1, resides within 200 bp of DSBs. Using laser microirradiation of cells, we demonstrate that pSp1 is present at DNA DSBs by 7.5 min after induction of damage and remains at the break site for at least 8 h. Depletion of Sp1 inhibits repair of site-specific DNA breaks, and the N-terminal 182-amino-acid peptide, which contains targets of ATM kinase but lacks the zinc finger DNA binding domain, is phosphorylated, localizes to DSBs, and rescues the repair defect resulting from Sp1 depletion. Together, these data demonstrate that Sp1 is rapidly recruited to the region immediately adjacent to sites of DNA DSBs and is required for DSB repair, through a mechanism independent of its sequence-directed transcriptional effects.