Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA.
Mol Cell Biol. 2012 Sep;32(18):3790-9. doi: 10.1128/MCB.00049-12. Epub 2012 Jul 23.
Sp1 is a ubiquitously expressed transcription factor that is phosphorylated by ataxia telangiectasia mutated kinase (ATM) in response to ionizing radiation and H(2)O(2). Here, we show by indirect immunofluorescence that Sp1 phosphorylated on serine 101 (pSp1) localizes to ionizing radiation-induced foci with phosphorylated histone variant γH2Ax 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.
Sp1 是一种普遍表达的转录因子，可被共济失调毛细血管扩张突变激酶 (ATM) 在受到电离辐射和 H₂O₂的刺激时磷酸化。在这里，我们通过间接免疫荧光显示，磷酸化丝氨酸 101 位的 Sp1（pSp1）与磷酸化组蛋白变体 γH2Ax 和 MRN（Mre11、Rad50 和 Nbs1）复合物的成员一起定位于电离辐射诱导的焦点中。通过染色质免疫沉淀（ChIP）对 DNA 双链断裂（DSBs）的占有率进行更精确的分析表明，Sp1 与 Nbs1 一样，位于 DSB 附近 200 bp 内。通过细胞的激光微照射，我们证明 pSp1 在诱导损伤后 7.5 分钟就存在于 DNA DSB 处，并至少在 8 小时内保持在断裂部位。Sp1 的耗竭抑制了特定部位 DNA 断裂的修复，而包含 ATM 激酶靶标的 N 端 182 个氨基酸肽（缺少锌指 DNA 结合结构域）被磷酸化，定位到 DSB 处，并挽救了 Sp1 耗竭导致的修复缺陷。总之，这些数据表明 Sp1 被迅速募集到 DNA DSB 附近的区域，并且是 DSB 修复所必需的，这一机制独立于其序列导向的转录效应。
