DNA REPAIR DEFECT IN FANCONI ANEMIA, GROUP A

本研究的目的是阐明Fanconi贫血患者的FAA和FAC基因产物与DNA修复缺陷之间的关系。有人假设，这种疾病的潜在机制可能涉及DNA修复缺陷。我们从FA-A和FA-C细胞的细胞核中分离出一个DNA内切酶复合体，并证明它在链间交联处切割DNA的能力是有缺陷的。在FA-A和FA-C细胞中，与该复合体相关并与交联型DNA结合的230 kDa蛋白质水平降低。该蛋白最近被鉴定为非红系阿尔法光谱蛋白(alphaSpIIsigma*)。在转导了表达FAA基因的逆转录病毒载体的FA-A细胞中，αSpIIsigma*的缺陷被纠正，表明FAA基因在其表达或稳定性方面发挥了作用。AlphaSpIIsigma*还与细胞核中的FAA和FAC蛋白形成复合体，提示该复合体可能在DNA修复中发挥作用。AlphaSpllsigma*可能作为一种支架，帮助对齐或增强参与链间交叉连接修复的蛋白质与与FAA和FAC相互作用的蛋白质之间的相互作用。目前的建议将通过首先确定我们已经鉴定的αSpllsigma*的异构体来解决这一问题，并生产一种可用于进一步研究的重组蛋白。究竟哪些蛋白质与FAA-FAC-alphaSpllsigma*复合体相关，这些蛋白质中是否有任何蛋白质与含有链间交联链的DNA具有结合亲和力，以及这些蛋白质中是否有任何蛋白质在FA-A和FA-C细胞中存在缺陷，将被确定。将评估FAA和FAC蛋白在调节α-Spllsigma表达或稳定性方面的作用，以及这三种蛋白在DNA链间交叉连接修复中的作用。如果AlphaSpllsigma*作为支架蛋白，帮助对齐并允许这些蛋白质和其他蛋白质之间的相互作用，这可能在许多不同的过程中产生深远的影响，除了与该蛋白质相关的DNA修复，如信号转导和细胞生长和发育。因此，FA细胞中α-Spllsigma*的缺失最终会影响造血的分化和发育。分离和鉴定与FAA-FAC-α-Spllsigma*复合体相关的蛋白质，并确定它们之间的相互作用、其他核蛋白和DNA修复，将有助于阐明FA骨髓衰竭的基础以及再生障碍性贫血和白血病的发展。

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.