FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ’s catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.

中文翻译：

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FANCJ 补偿 RAP80 缺陷并抑制由链间交联引起的基因组不稳定性。

FANCJ 是一种 DNA 解旋酶，是肿瘤抑制因子 BRCA1 的相互作用伙伴，对于修复 DNA 链间交联 (ICL) 至关重要，ICL 是一种导致染色体不稳定和干扰正常转录的剧毒病变。在二倍体细胞中，FANCJ 被认为在 DNA 双链断裂 (DSB) 的同源重组 (HR) 修复中起作用；然而，人们对其确切的作用和分子机制知之甚少。此外，尚未探索 FANCJ 缺陷时 ICL 抵抗的代偿机制。在这项工作中，我们进行了 siRNA 筛选以确定 DNA 损伤反应/DNA 修复机制的基因，当急性耗尽时，FANCJ CRISPR 敲除细胞对低浓度的 DNA 交联剂丝裂霉素 C (MMC) 敏感。屏幕上的热门歌曲之一是 RAP80，一种蛋白质，它招募修复机制来破坏 DNA 末端并调节 DNA 末端加工。FANCJ 和 RAP80 的伴随缺失不仅会加重人类细胞中的 DNA 损伤水平，还会对细胞周期检查点产生不利影响，从而导致染色体严重不稳定。遗传互补实验表明，当 RAP80 缺乏时，FANCJ 的催化活性和与 BRCA1 的相互作用对 ICL 抗性很重要。暴露于 MMC 的 FANCJ 和 RAP80 共同缺乏的细胞中 RPA 和 RAD51 病灶升高归因于 Mre11 和 CtIP 核酸酶产生的单链 DNA。总之，我们基于细胞的研究结果以及生化研究表明，FANCJ 在 ICL 诱导的 DNA 损伤修复过程中具有抑制不完全加工和有毒的关节 DNA 分子的关键功能。