Abstract：
BACKGROUND: Sn1-type alkylating agents methylate the oxygen atom on guanine bases thereby producing O6-methylguanine. This modified base could pair with thymine and cytosine, resulting in the formation of O6-methylguanine/thymine mismatch during DNA replication, recognized by the mismatch repair (MMR) complex, which then initiates the DNA damage response and subsequent apoptotic processes. In our investigation of the molecular mechanisms underlying MMR-dependent apoptosis, we observed FANCD2 modification upon the activity of alkylating agent N-methyl-N-nitrosourea (MNU). This observation led us to hypothesize a relevant role for FANCD2 in the apoptosis induction process.
RESULTS: We generated FANCD2 knockout cells using the CRISPR/Cas9 method in the human cervical cancer cell line HeLa MR. FANCD2-deficient cells exhibited MNU hypersensitivity. Upon MNU exposure, FANCD2 colocalized with the MMR complex. MNU-treated FANCD2 knockout cells displayed severe S phase delay followed by increased G2/M arrest and MMR-dependent apoptotic cell death. Moreover, FANCD2 knockout cells exhibited impaired CtIP and RAD51 recruitment to the damaged chromatin and DNA double-strand break accumulation, indicated by simultaneously observed increased γH2AX signal and 53BP1 foci.
CONCLUSIONS: Our data suggest that FANCD2 is crucial for recruiting homologous recombination factors to the sites of the MMR-dependent replication stress to resolve the arrested replication fork and counteract O6-methylguanine-triggered MMR-dependent apoptosis.
RESULTS: We generated FANCD2 knockout cells using the CRISPR/Cas9 method in the human cervical cancer cell line HeLa MR. FANCD2-deficient cells exhibited MNU hypersensitivity. Upon MNU exposure, FANCD2 colocalized with the MMR complex. MNU-treated FANCD2 knockout cells displayed severe S phase delay followed by increased G2/M arrest and MMR-dependent apoptotic cell death. Moreover, FANCD2 knockout cells exhibited impaired CtIP and RAD51 recruitment to the damaged chromatin and DNA double-strand break accumulation, indicated by simultaneously observed increased γH2AX signal and 53BP1 foci.
CONCLUSIONS: Our data suggest that FANCD2 is crucial for recruiting homologous recombination factors to the sites of the MMR-dependent replication stress to resolve the arrested replication fork and counteract O6-methylguanine-triggered MMR-dependent apoptosis.
摘要：
背景:Sn1型烷基化剂将鸟嘌呤碱基上的氧原子甲基化,从而产生O6-甲基鸟嘌呤。这个修饰的碱基可以与胸腺嘧啶和胞嘧啶配对，导致在DNA复制过程中形成O6-甲基鸟嘌呤/胸腺嘧啶错配，由错配修复(MMR)复合物识别，然后启动DNA损伤反应和随后的凋亡过程。在我们对MMR依赖性细胞凋亡的分子机制的研究中，我们观察到FANCD2修饰对烷化剂N-甲基-N-亚硝基脲（MNU）活性的影响。这一观察结果使我们假设FANCD2在凋亡诱导过程中的相关作用。
结果：我们使用CRISPR/Cas9方法在人宫颈癌细胞系HeLaMR中产生了FANCD2敲除细胞。FANCD2缺陷型细胞表现出MNU超敏反应。MNU暴露后，FANCD2与MMR复合物共定位。MNU处理的FANCD2敲除细胞表现出严重的S期延迟，随后G2/M阻滞和MMR依赖性凋亡细胞死亡增加。此外，FANCD2敲除细胞表现出受损的CtIP和RAD51募集到受损的染色质和DNA双链断裂积累，同时观察到增加的γH2AX信号和53BP1病灶。
结论：我们的数据表明，FANCD2对于招募同源重组因子到MMR依赖性复制应激位点以解决停滞的复制叉并抵消O6-甲基鸟嘌呤触发的MMR依赖性凋亡至关重要。
结果：我们使用CRISPR/Cas9方法在人宫颈癌细胞系HeLaMR中产生了FANCD2敲除细胞。FANCD2缺陷型细胞表现出MNU超敏反应。MNU暴露后，FANCD2与MMR复合物共定位。MNU处理的FANCD2敲除细胞表现出严重的S期延迟，随后G2/M阻滞和MMR依赖性凋亡细胞死亡增加。此外，FANCD2敲除细胞表现出受损的CtIP和RAD51募集到受损的染色质和DNA双链断裂积累，同时观察到增加的γH2AX信号和53BP1病灶。
结论：我们的数据表明，FANCD2对于招募同源重组因子到MMR依赖性复制应激位点以解决停滞的复制叉并抵消O6-甲基鸟嘌呤触发的MMR依赖性凋亡至关重要。
