Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, OH, USA.
Department of Pediatrics and Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Nucleic Acids Res. 2019 Apr 23;47(7):3503-3520. doi: 10.1093/nar/gkz026.
The primary function of the UBE2T ubiquitin conjugase is in the monoubiquitination of the FANCI-FANCD2 heterodimer, a central step in the Fanconi anemia (FA) pathway. Genetic inactivation of UBE2T is responsible for the phenotypes of FANCT patients; however, a FANCT patient carrying a maternal duplication and a paternal deletion in the UBE2T loci displayed normal peripheral blood counts and UBE2T protein levels in B-lymphoblast cell lines. To test whether reversion by recombination between UBE2T AluYa5 elements could have occurred in the patient's hematopoietic stem cells despite the defects in homologous recombination (HR) in FA cells, we constructed HeLa cell lines containing the UBE2T AluYa5 elements and neighboring intervening sequences flanked by fluorescent reporter genes. Introduction of a DNA double strand break in the model UBE2T locus in vivo promoted single strand annealing (SSA) between proximal Alu elements and deletion of the intervening color marker gene, recapitulating the reversion of the UBE2T duplication in the FA patient. To test whether UBE2T null cells retain HR activity, the UBE2T genes were knocked out in HeLa cells and U2OS cells. CRISPR/Cas9-mediated genetic knockout of UBE2T only partially reduced HR, demonstrating that UBE2T-independent pathways can compensate for the recombination defect in UBE2T/FANCT null cells.
UBE2T 泛素连接酶的主要功能是单泛素化 FANCI-FANCD2 异二聚体，这是范可尼贫血（FA）途径中的一个关键步骤。UBE2T 的遗传失活导致 FANCT 患者出现表型；然而，一位携带 UBE2T 基因座母源性重复和父源性缺失的 FANCT 患者在 B 淋巴细胞系中显示出正常的外周血计数和 UBE2T 蛋白水平。为了测试在 FA 细胞中同源重组（HR）缺陷的情况下，UBE2T AluYa5 元件之间的重组是否可以在患者的造血干细胞中发生逆转，我们构建了含有 UBE2T AluYa5 元件和侧翼带有荧光报告基因的邻近间隔序列的 HeLa 细胞系。在体内模型 UBE2T 基因座中引入 DNA 双链断裂促进了近端 Alu 元件之间的单链退火（SSA）和间隔彩色标记基因的缺失，重现了 FA 患者中 UBE2T 重复的逆转。为了测试 UBE2T 缺失细胞是否保留 HR 活性，我们在 HeLa 细胞和 U2OS 细胞中敲除了 UBE2T 基因。CRISPR/Cas9 介导的 UBE2T 基因敲除仅部分降低了 HR，表明 UBE2T 非依赖性途径可以补偿 UBE2T/FANCT 缺失细胞的重组缺陷。
