Regulation of the Fanconi Anemia Pathway by Ubiquitination

范可尼贫血（FA）是一种常染色体隐性遗传的癌症易感性疾病，其特征在于先天性 异常、骨髓衰竭和对DNA交联剂的细胞超敏性。九个FA基因 已被克隆，并且九种编码的蛋白质（A、B、C、D1、D2、E、F、G、L）以共同的 细胞途径，导致FANCD 2的单泛素化及其在染色质中与 FANCD1/BRCA 2.有趣的是，一种新的去泛素化酶，称为USP 1， 并关闭该途径1（Nijman等，Molecular Cell 17：331，2005）。人们对这项规定知之甚少 USP 1、USP 1的其他底物或USP 1在DNA修复中的整体细胞功能。最近， 我们对USP 1有两个重要的观察。首先，USP 1在DNA降解后迅速降解， 损伤，导致FANCD 2-Ub的积累。这一结果表明FANCD 2-Ub的积累 这是由于FA复合物减少去泛素化和增加泛素化的结果。二是 除了FANCD 2-Ub，USP 1还使持续合成因子PCNA去泛素化，从而调节 跨损伤DNA合成（TLS）。这一观察结果表明，FA之间存在功能性联系， 途径和TLS中变异DNA聚合酶的DNA复制。根据这些新的观察，我们 提出今后五年的三个奋斗目标。在具体目标#1中，我们将检查分子 DNA损伤诱导的USP 1降解机制。在具体目标#2中，我们将检查细胞 USP 1失调的结果（即对FA途径介导和TLS介导的DNA修复的影响） 活性和突变频率）。为此，我们将使用新生成的Usp 1缺陷小鼠模型。 在具体目标#3中，我们将研究FANCD 2突变体的单泛素化和去泛素化， 在体外和体内的多肽。我们还将确定这些FANCD 2突变蛋白的相互作用， FA复合物和USP 1。总的来说，我们相信这些研究将阐明细胞调节 控制关键生物调节剂如FANCD 2的单泛素化状态的机制。

Fanconi Anemia (FA) is an autosomal recessive cancer susceptibility disorder characterized by congenital abnormalities, bone marrow failure, and cellular hypersensitivity to DNA crosslinking agents. Nine FA genes have been cloned, and the nine encoded proteins (A, B, C, D1, D2, E, F, G, L) cooperate in a common cellular pathway, leading to the monoubiquitination of FANCD2 and its interaction in chromatin with FANCD1/BRCA2. Interestingly, a novel deubiquitinating enzyme, called USP1, deubiquitinates FANCD2 and shuts the pathway "off1 (Nijman et al, Molecular Cell 17: 331,2005). Little is known about the regulation of USP1, the other substrates of USP1, or the global cellular function of USP1 in DNA repair. Very recently, we have made two important observations about USP1. First, USP1 is rapidly degraded following DNA damage, resulting in the accumulation of FANCD2-Ub. This result suggests that FANCD2-Ub accumulation results from decreased deubiquitination as well as increased ubiquitination by the FA complex. Second, in addition to FANCD2-Ub, USP1 also deubiquitinates the processivity factor, PCNA, and thus regulates translesion DNA synthesis (TLS). This observation suggests a functional connection between the FA pathway and DNA replication by variant DNA polymerases in TLS. Based on these new observations, we propose the following three aims for the next five years. In Specific Aim #1, we will examine the molecular mechanism of DNA damage-inducible USP1 degradation. In Specific Aim #2, we will examine the cellular outcome of USP1 dysregulation (namely, the affect on FA pathway mediated and TLS-mediated DNA repair activity and mutation frequency). For this aim, we will use a newly-generated Usp1-deficient mouse model. In Specific Aim #3, we will examine the monoubiquitination and deubiquitination of FANCD2 mutant polypeptides in vitro and in vivo. We will also determine the interaction of these FANCD2 mutant proteins with the FA complex and with USP1. Overall, we believe these studies will elucidate the cellular regulatory mechanisms which control the monoubiquitination state of key biological modulators, such as FANCD2.