在链间交联(ICL)的修复期间,产生DNA双链断裂(DSB)。范可尼贫血(FA)核心复合物,被招募到ICL,通过同源重组(HR)促进该DSB的高保真修复。然而,FA核心复合体是否也促进了独立于ICL的DSB的HR,例如由电离辐射或核酸酶诱导,仍然有争议。这里,在基于CRISPR/Cas9的筛选中,我们将FA核心复合物成员FANCL和Ube2T确定为HR促进因子.使用等基因细胞系模型,我们进一步证明了FANCL和Ube2T的HR促进功能,和它们的泛素化底物FANCD2。我们证明了FANCL和Ube2T以依赖于FANCM的方式定位在DSB,并且是FANCD2的DSB积累所必需的。机械上,我们证明FANCL泛素连接酶活性是CtIP在DSB的积累所必需的,从而促进末端切除和Rad51加载。一起,这些数据表明FA核心复合物和FANCD2在促进ICL和DSB修复中具有双重基因组维持功能.
During the repair of interstrand crosslinks (ICLs) a DNA double-strand break (DSB) is generated. The Fanconi anemia (FA) core complex, which is recruited to ICLs, promotes high-fidelity repair of this DSB by homologous recombination (HR). However, whether the FA core complex also promotes HR at ICL-independent DSBs, for example induced by ionizing irradiation or nucleases, remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen. Using isogenic cell line models, we further demonstrated an HR-promoting function of FANCL and Ube2T, and of their ubiquitination substrate FANCD2. We show that FANCL and Ube2T localize at DSBs in a FANCM-dependent manner, and are required for the DSB accumulation of FANCD2. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of CtIP at DSBs, thereby promoting end resection and Rad51 loading. Together, these data demonstrate a dual genome maintenance function of the FA core complex and FANCD2 in promoting repair of both ICLs and DSBs.