DNA REPAIR DEFECT IN FANCONI ANEMIA, GROUP A

本提案的目的是描述FAA和FAC基因产物与Fanconi贫血，补体组A （FA-A）和C (FA-C) DNA修复缺陷之间的关系。据推测，这种疾病的潜在机制可能与DNA修复缺陷有关。我们从FA-A和FA-C细胞的细胞核中分离出一种DNA内切酶复合体，并表明它在链间交联位点切割DNA的能力有缺陷。在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蛋白水平降低。该蛋白最近被鉴定为非红系α spectrinllsigma* (alphaSpIIsigma*)。用表达FAA cDNA的逆转录病毒载体转导FA-A细胞，纠正了alphaSpIIsigma*的缺陷，表明FAA基因在其表达或稳定性中起作用。alphaSpIIsigma*也在细胞核中与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*可能作为支架，帮助排列或增强参与链间交联修复的蛋白和与FAA和FAC相互作用的蛋白之间的相互作用。目前的提案将通过首先确定我们已经确定的alphaSpllsigma*的异构体并产生可用于进一步研究的重组蛋白来解决这个问题。确切地说，哪些蛋白质与fa - fa - alphaspllsigma *复合物相关，这些蛋白质是否与含有链间交联的DNA具有结合亲和力，以及FA-A和FA-C细胞中是否存在这些蛋白质的缺乏将被确定。将评估FAA和FAC蛋白在调节alphaSpllsigma*的表达或稳定性中的作用，以及这三种蛋白在DNA链间交联修复中的作用。如果alphaSpllsigma*作为一种支架蛋白，帮助排列并允许这些蛋白和其他蛋白之间的相互作用，这可能对许多不同的过程产生深远的影响，除了DNA修复，这与该蛋白有关，如信号转导和细胞生长和发育。因此，FA细胞中alphaSpllsigma*的缺乏最终会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，并确定它们之间、其他核蛋白和DNA修复之间的相互作用，将有助于阐明FA患者骨髓衰竭和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.