Functional links between Fanconi anemia proteins and interstrand crosslinks

范可尼贫血（FA）是一种通过常染色体隐性遗传或x连锁模式遗传的遗传性疾病。FA患者表现为进行性骨髓功能障碍、全血细胞减少和急剧升高的癌症易感性。已经鉴定出13种不同的范可尼基因（FANCA - I）的种系突变，每种突变对应于一个不同的互补组。Fanconi患者细胞的标志是对双功能烷基化剂的敏感性显著增加，这些双功能烷基化剂能够形成DNA链间交联，这表明Fanconi通路在DNA损伤反应中起重要作用。与这一观点一致的是，FANC蛋白的功能似乎围绕着DNA损伤依赖性的FANCD2单泛素化进行组织。FANCI被发现是ATM/ATR检查点信号起始激酶的直接底物。然而，FANC基因产物如何帮助保护细胞免受交联DNA的伤害仍然是一个关键问题。项目3的目标是研究招募到DNA链间交联位点的蛋白质，并阐明它们在交联修复和损伤反应信号传导中的作用。我们的假设是，范可尼贫血途径的成分参与交联损伤反应的不同方面。这一假设是由三个具体目标来解决的。在目的1和2中，我们将使用一种新的方法来研究范可尼贫血途径的组成部分，以DNA复制依赖和独立的方式将其招募到交联位点。已被认为参与ICL加工的候选蛋白也将检查其在病变部位的存在。最后，我们将尝试一种无偏的纯化方法，旨在识别交联处理中涉及的新因素。这些研究有望为理解Fanconi贫血途径以及ICL修复机制提供重要的见解。

Fanconi anemia (FA) is a genetic disorder inherited via autosomal recessive or X-linked patterns. FA patients manifest progressive bone marrow dysfunction, pancytopenia, and drastically elevated cancer predisposition. Germline mutations in 13 different Fanconi genes (FANCA - I) have been identified, each corresponding to a distinct complementation group. The hallmark of Fanconi patient cells is profoundly increased sensitivity to bifunctional alkylating agents capable of forming DNA interstrand crosslinks, suggesting an important role of the Fanconi pathway in DNA damage response. Consistent with this notion, functions of the FANC proteins seems to organize around the DNA damage-dependent monoubiquitination of FANCD2. FANCI was found to be a direct substrate of the ATM/ATR checkpoint signal initiation kinases. However, how FANC gene products help protecting cells from with crosslinked DNA remains a key question. The goal of Project 3 is to study proteins recruited to the site of DNA interstrand crosslinks and to elucidate their role in crosslink repair and damage response signaling. We hypothesis is that components of the Fanconi anemia pathway is involved in different aspects of responses to crosslinking damage. This is hypothesis is addressed by the three Specific Aims. In Aim 1 and 2, we will use a novel approach to investigate components of the Fanconi anemia pathway for its ability to be recruited to the site of crosslink in a DNA replication-dependent and -independent manner. Candidate proteins that have been suggested to have involvement in ICL processing will also be examining for their presence at the site of the lesion. Finally, we will attempt an unbiased purification approach aimed at identify novel factor involved in crosslink processing. These studies are expected to provide significant insights toward the understanding of the Fanconi anemia pathway as well as mechanisms of ICL repair.