DNA Repair Defect in Fanconi Anemia, Group A

描述（由申请人提供）：范可尼贫血（FA）患者的细胞对DNA链间交联剂的致裂性和细胞毒性作用的超敏感性，以及它们修复这些药物造成的损伤的能力缺陷，导致了这种疾病的发病机制涉及DNA修复缺陷的假设。本提案的目标是描述FANC蛋白与那些参与修复过程的关键、初始损伤识别和切口步骤的蛋白之间的关系，并确定它们的功能重要性。我们已经鉴定出一种结构蛋白，非红系α谱蛋白（alphaSpII-sigma*）是正常细胞核中蛋白质复合物的组成部分，并表明它与交联DNA结合，并且在所有FA细胞系中都缺乏。这种缺陷在表达适当的FANC cdna的FA-A、FA-C和FA-G细胞中得到纠正，表明FANC蛋白参与其中。我们假设alphaSpII-sigma*是FA细胞修复缺陷的关键因素，它作为支架帮助在损伤部位招募修复蛋白，帮助它们对齐和相互作用，并且alphaSpII-sigma*与FANC蛋白的相互作用对修复过程至关重要。为了解决这一假设，我们将在正常细胞中进行sirna介导的alphaSpII-sigma*和FANC表达的沉默，以确定这些基因在修复过程中的功能重要性。FANC和DNA修复蛋白是否与alphaSpII-sigma*在细胞核交联位点共定位将在光镜和电镜水平上进行检查，并确定这种共定位的动力学。酵母双杂交分析将用于确定alphaSpII-sigma*、fnc蛋白和参与交联修复的DNA修复蛋白之间是否存在直接相互作用，并确定这些相互作用所涉及的结构域。这些蛋白质相互作用的动力学将被研究。此外，将确定fnc蛋白是否直接与交联DNA结合，如果是，将检查这些蛋白和alphaSpII-sigma*上的特定结合域。这些研究将阐明alphaSpII-sigma*和FANC蛋白在DNA修复中的作用，以及alphaSpII-sigma*与FANC蛋白相互作用对其稳定性的重要性。由于除了DNA修复外，aII谱蛋白还与细胞中的许多不同过程有关，例如信号转导和细胞生长和分化，因此FA细胞中alphaSpII-sigma*的缺乏可能会产生深远的影响。因此，阐明alphaSpII-sigma*与fnc蛋白之间的关系，可能为FA中有缺陷的造血分化和发育，以及再生障碍性贫血、白血病和其他癌症提供潜在的基础。

DESCRIPTION (provided by applicant): Hypersensitivity of cells from patients with Fanconi anemia (FA) to the clastogenic and cytotoxic effects of DNA interstrand cross-linking agents and their defect in ability to repair damage produced by these agents has led to the hypothesis that the etiopathogenesis of this disorder involves a DNA repair defect. The goals of this proposal are to delineate the relationship between the FANC proteins and those proteins involved in the critical, initial damage recognition and incision steps of the repair process and to ascertain their functional importance. We have identified a structural protein, nonerythroid alpha spectrin (alphaSpII-sigma*) as a component of a protein complex in the nucleus of normal cells and shown that it binds to cross-linked DNA and is deficient in all FA cell lines tested. This deficiency is corrected in FA-A, FA-C and FA-G cells expressing the appropriate FANC cDNAs, indicating involvement of the FANC proteins. We have hypothesized that alphaSpII-sigma* is a critical factor in the repair defect in FA cells, that it acts as a scaffold to help recruit repair proteins at sites of damage, aiding in their alignment and interactions, and that the interaction of alphaSpII-sigma* with the FANC proteins is essential for the repair process. To address this hypothesis, siRNA-mediated silencing of alphaSpII-sigma* and FANC expression in normal cells will be carried out to determine the functional importance of these genes in the repair process. Whether the FANC and DNA repair proteins co-localize with alphaSpII-sigma* at sites of cross-links in the nucleus will be examined at both the light and electron microscopic levels and the kinetics of this co-localization ascertained. Yeast-two-hybrid analysis will be used to determine whether there are direct interactions between alphaSpII-sigma*, the FANC proteins and DNA repair proteins involved in cross-link repair and ascertain the domains involved in these interactions. The kinetics of these protein interactions will be studied. Also, whether the FANC proteins bind directly to cross-linked DNA will be determined and, if so, the specific binding domains on these proteins and on alphaSpII-sigma* will be examined. These studies should elucidate the role of alphaSpII-sigma* and the FANC proteins in DNA repair and the importance of the interaction of alphaSpII-sigma* with the FANC proteins for its stability. Since aII spectrin has been associated with a number of different processes in the cell besides DNA repair, such as signal transduction and cell growth and differentiation, a deficiency in alphaSpII-sigma* in FA cells could have far reaching consequences. Thus elucidating the relationship between alphaSpII-sigma* and the FANC proteins could potentially delineate the basis for defective hematopoietic differentiation and development, and for aplastic anemia, leukemia and other cancers in FA.