背景:转换/蔗糖不可发酵(SWI/SNF)相关,关联矩阵,染色质的肌动蛋白依赖性调节剂,亚家族A(SMARCA)成员2和成员4(SMARCA2/4)是旁系同源物,并且充当用于染色质重塑的SWI/SNF复合物中的关键酶亚基。然而,SMARCA2/4在DNA损伤应答中的作用尚不清楚.
方法:进行激光微照射试验以检查SMARCA2/4的关键结构域,以将SWI/SNF复合物重新定位到DNA损伤中。为了检查介导SMARCA2/4募集的关键因素,在用共济失调毛细血管扩张突变(ATM)抑制剂处理的HeLa细胞中检查了SMARCA2/4向DNA损伤的重新定位。共济失调毛细血管扩张症和Rad3相关蛋白(ATR),CREB结合蛋白(CBP)及其同源物p300(p300/CBP),或聚(ADP-核糖)聚合酶(PARP)1/2以及H2AX缺陷型HeLa细胞。此外,通过用小分子抑制剂FHD286或化合物14伴随抑制SMARCA2/4,检查了SMARCA2/4在辐射敏感51(RAD51)病灶形成和同源重组修复中的功能。最后,使用集落形成测定法,研究了PARP抑制剂和SMARCA2/4抑制剂对抑制肿瘤细胞生长的协同作用。
结果:我们表明SMARCA2/4在DNA损伤后重新定位到DNA损伤,这需要他们的ATP酶活性。此外,这些ATP酶活性也是SWI/SNF复合物中其他亚基向DNA损伤转移所必需的.有趣的是,SMARCA2/4的重新定位独立于γH2AX,ATM,ATR,p300/CBP,或PARP1/2,表明它可以直接识别DNA损伤作为DNA损伤传感器。缺乏SMARCA2/4延长了γH2AX的保留,环指蛋白8(RNF8)和乳腺癌易感基因1(BRCA1)在DNA损伤和损害RAD51依赖性同源重组修复。此外,SMARCA2/4抑制剂的治疗使肿瘤细胞对PARP抑制剂治疗敏感。
结论:本研究揭示SMARCA2/4作为双链断裂修复的DNA损伤修复因子。
BACKGROUND: The switching/sucrose non-fermentable (SWI/SNF) Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A (SMARCA) member 2 and member 4 (SMARCA2/4) are paralogs and act as the key enzymatic subunits in the SWI/SNF complex for chromatin remodeling. However, the role of SMARCA2/4 in DNA damage response remains unclear.
METHODS: Laser microirradiation assays were performed to examine the key domains of SMARCA2/4 for the relocation of the SWI/SNF complex to DNA lesions. To examine the key factors that mediate the recruitment of SMARCA2/4, the relocation of SMARCA2/4 to DNA lesions was examined in HeLa cells treated with inhibitors of Ataxia-telangiectasia-mutated (ATM), Ataxia telangiectasia and Rad3-related protein (ATR), CREB-binding protein (CBP) and its homologue p300 (p300/CBP), or Poly (ADP-ribose) polymerase (PARP) 1/2 as well as in H2AX-deficient HeLa cells. Moreover, by concomitantly suppressing SMARCA2/4 with the small molecule inhibitor FHD286 or Compound 14, the function of SMARCA2/4 in Radiation sensitive 51 (RAD51) foci formation and homologous recombination repair was examined. Finally, using a colony formation assay, the synergistic effect of PARP inhibitors and SMARCA2/4 inhibitors on the suppression of tumor cell growth was examined.
RESULTS: We show that SMARCA2/4 relocate to DNA lesions in response to DNA damage, which requires their ATPase activities. Moreover, these ATPase activities are also required for the relocation of other subunits in the SWI/SNF complex to DNA lesions. Interestingly, the relocation of SMARCA2/4 is independent of γH2AX, ATM, ATR, p300/CBP, or PARP1/2, indicating that it may directly recognize DNA lesions as a DNA damage sensor. Lacking SMARCA2/4 prolongs the retention of γH2AX, Ring Finger Protein 8 (RNF8) and Breast cancer susceptibility gene 1 (BRCA1) at DNA lesions and impairs RAD51-dependent homologous recombination repair. Furthermore, the treatment of an SMARCA2/4 inhibitor sensitizes tumor cells to PARP inhibitor treatment.
CONCLUSIONS: This study reveals SMARCA2/4 as a DNA damage repair factor for double-strand break repair.