Seki Sohsuke, Ohzeki Mioko, Uchida Akiko, Hirano Seiki, Matsushita Nobuko, Kitao Hiroyuki, Oda Tsukasa, Yamashita Takayuki, Kashihara Naoki, Tsubahara Akio, Takata Minoru, Ishiai Masamichi
Department of Immunology and Molecular Genetics, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan.
Genes Cells. 2007 Mar;12(3):299-310. doi: 10.1111/j.1365-2443.2007.01054.x.
The rare hereditary disorder Fanconi anemia (FA) can be caused by mutations in components of the FA core complex (FancA/B/C/E/F/G/L/M), a key regulator FancD2, the breast cancer susceptibility protein BRCA2/FancD1, or the newly identified FancJ/BRIP1 helicase. By performing yeast two-hybrid (Y2H) screens using N-terminal chicken (ch) FancD2 as a bait, we have identified chFancL, the likely ubiquitin E3 ligase subunit of the FA core complex. We also found that ectopically expressed FancD2 and FancL co-immunoprecipitated in 293T cells, and this interaction was dependent on the PHD domain of FancL. FANCL-disrupted chicken DT40 cells displayed defects in both FancD2 monoubiquitination and focus formation. Importantly, cell lines lacking the FANCL or FANCD2 genes, or carrying a "knock-in" mutation of the FancD2 monoubiquitination site (where the Lys 563 residue is changed to Arg), displayed quantitatively identical defects in the repair of I-SceI-induced chromosomal breaks by homologous recombination (HR). These data establish the role of FANCL and FancD2 monoubiquitination in HR repair.
罕见的遗传性疾病范可尼贫血（FA）可能由FA核心复合物（FancA/B/C/E/F/G/L/M）的组分、关键调节因子FancD2、乳腺癌易感蛋白BRCA2/FancD1或新发现的FancJ/BRIP1解旋酶发生突变引起。通过以N端鸡（ch）FancD2作为诱饵进行酵母双杂交（Y2H）筛选，我们鉴定出chFancL，它可能是FA核心复合物的泛素E3连接酶亚基。我们还发现，异位表达的FancD2和FancL在293T细胞中共免疫沉淀，且这种相互作用依赖于FancL的PHD结构域。FANCL缺失的鸡DT40细胞在FancD2单泛素化和灶形成方面均表现出缺陷。重要的是，缺乏FANCL或FANCD2基因，或携带FancD2单泛素化位点“敲入”突变（其中赖氨酸563残基变为精氨酸）的细胞系，在通过同源重组（HR）修复I-SceI诱导的染色体断裂方面表现出数量上相同的缺陷。这些数据确立了FANCL和FancD2单泛素化在HR修复中的作用。
