DNA REPAIR DEFECT IN FANCONI ANEMIA, GROUP A

本研究的目的是阐明Fanconi贫血中FAA和FAC基因产物与DNA修复缺陷之间的关系，互补组A（FA-A）和C（FA-C）。据推测，这种疾病的潜在机制可能涉及DNA修复缺陷。我们已经从FA-A和FA-C细胞的细胞核中分离出了DNA内切酶复合物，并表明它在链间交联位点切割DNA的能力上是有缺陷的。FA-A和FA-C细胞中与该复合物相关并与交联DNA结合的230 kDa蛋白质的水平降低。这种蛋白质最近被鉴定为非红细胞α血影蛋白σ *（alphaSpIIsigma*）。在用表达FAA cDNA的逆转录病毒载体转导的FA-A细胞中，α SpII sigma * 的缺陷得到纠正，表明FAA基因在其表达或稳定性中起作用。alphaSpII sigma * 还与细胞核中的FAA和FAC蛋白形成复合物，这表明该复合物可能在DNA修复中起作用。alphaSpllsigma* 有可能作为支架，帮助对齐或增强参与修复链间交联的蛋白质与与FAA和FAC相互作用的蛋白质之间的相互作用。本提案将通过首先确定我们已经鉴定的alphaSpllsigma* 的同种型并产生可用于进一步研究的重组蛋白来解决这一问题。将确定确切地哪些蛋白质与FAA-FAC-alphaSpllsigma* 复合物相关，这些蛋白质中的任一种是否对含有链间交联的DNA具有结合亲和力，以及FA-A和FA-C细胞中这些蛋白质中的任一种是否存在缺陷。将评估FAA和FAC蛋白在调节alphaSpllsigma* 的表达或稳定性中的作用，以及这三种蛋白中的每一种在修复DNA链间交联中的作用。如果alphaSpllsigma* 作为支架蛋白，帮助这些蛋白质以及其他蛋白质之间的对齐和相互作用，这可能对许多不同的过程产生深远的影响，除了DNA修复，这些过程与这种蛋白质有关，如信号转导和细胞生长和发育。因此，FA细胞中的alphaSpllsigma* 缺陷可能最终影响造血分化和发育。分离和鉴定与FAA-FAC- alphaSpllsigma* 复合物相关的蛋白质，并确定它们彼此之间、与其他核蛋白之间以及与DNA修复之间的相互作用，这将有助于阐明FA中骨髓衰竭以及再生障碍性贫血和白血病发生的基础。

The goal of this proposal is to delineate the relationship between the FAA and FAC gene products and the DNA repair defect in Fanconi anemia, complementation groups A (FA-A) and C (FA-C). It has been hypothesized that an underlying mechanism for this disorder may involve a DNA repair defect. We have isolated a DNA endonuclease complex from the nuclei of FA-A and FA-C cells and shown that it is defective in ability to incise DNA at sites of interstrand cross- links. Levels of a 230 kDa protein, associated with this complex and which binds to cross-linked DNA, are decreased in FA-A and FA-C cells. This protein has recently been identified as nonerythroid alpha spectrinllsigma* (alphaSpIIsigma*). The deficiency in alphaSpIIsigma* is corrected in FA-A cells transduced with a retroviral vector expressing the FAA cDNA, indicating that the FAA gene plays a role in its expression or stability. alphaSpIIsigma* also forms a complex with the FAA and FAC proteins in the nucleus which suggests that this complex may play role in DNA repair. It is possible that alphaSpllsigma* acts as a scaffold to help align or enhance interaction between proteins involved in the repair of interstrand cross-links and proteins that interact with FAA and FAC. The present proposal will address this by first determining the isoform of the alphaSpllsigma* we have identified and producing a recombinant protein that can be used in further studies. Exactly what proteins are associated with the FAA-FAC-alphaSpllsigma* complex, whether any of these proteins have binding affinity for DNA containing interstrand cross-links, and whether there is a deficiency in any of these proteins in FA-A and FA-C cells will be determined. The role of the FAA and FAC proteins in regulating the expression or stability of alphaSpllsigma* will be assessed as will the role of each of these three proteins in the repair of DNA interstrand cross-links. If alphaSpllsigma* is acting as a scaffolding protein, to help align and allow interactions between these as well as other proteins, this could have far reaching implications in a number of different processes, in addition to DNA repair, which have been associated with this protein, such as signal transduction and cell growth and development. A deficiency in alphaSpllsigma* in FA cells could thus ultimately affect hematopoietic differentiation and development. Isolation and identification of proteins associated with the FAA-FAC- alphaSpllsigma* complex and determination of their interactions with each other, other nuclear proteins, and DNA repair should help elucidate the basis of bone marrow failure and the development of aplastic anemia and leukemia in FA.