Functional analysis of the FANCD2 interactors containing ubiquitin binding domain

Fanconi贫血(FA)途径在DNA损伤反应和DNA修复中起重要作用。FANCD2是一种关键蛋白，它是一种单体蛋白，在DNA链间交联(ICL)的作用下聚集在染色质中，通过目前尚不清楚的机制协调DNA修复。我们已经分离到UBR5和UFD1作为FANCD2的候选相互作用蛋白。这两种蛋白都含有泛素结合域。我们已经建立了ubr5缺失的鸡DT40细胞，以及基于siRNA的ufd1-敲除人类细胞。出乎意料的是，与野生型细胞相比，ubr5缺陷或ufd1基因敲除的细胞对ICL试剂的敏感性很低。用表达GFP-UBR5或GFP-UFD1的人细胞进行激光微照射实验表明，DNA损伤后GFP蛋白几乎没有积聚。这些结果表明，至少在ICL修复过程中，FA通路与UBR5或包括相关蛋白的UFD1之间的功能关系不强。

The Fanconi anemia (FA) pathway is critically involved in DNA damage responses and DNA repair. A key protein, FANCD2, is monoubiquitinated and accumulates in chromatin in response to DNA interstrand crosslinks (ICLs), where it coordinates DNA repair through mechanisms that are still poorly understand.We have isolated UBR5 and UFD1 as candidate interacting proteins for FANCD2. Both proteins contain ubiquitin-binding domains. We have established ubr5-deficient chicken DT40 cells, and SiRNA-based ufd1-knock down human cells. Unexpectedly, ubr5-deficient or ufd1-knock down cells showed little sensitivity to ICL reagents compared to wild type cells. Laser microirradiation experiments using GFP-UBR5 or GFP-UFD1 expressing human cells showed little GFP-protein accumulation after DNA damage. These results suggested that functional relationship between FA pathway and UBR5, or UFD1 including related proteins was not strong, at least in ICL repair.