Sadofsky-RAG1 N-terminal domain binding interactions

描述（由申请人提供）：脊椎动物的适应性免疫系统依赖于DNA重组系统，称为V(D)J重组，以产生免疫球蛋白和T细胞受体。当误用时，这一重要步骤也会导致染色体易位。这种重组过程的大部分调控发生在DNA被切割的初始阶段。蛋白质RAG1和RAG2形成位点特异性核酸酶，切割合适的DNA靶标，并被认为协调重组反应。RAG1蛋白具有一个大的n端结构域，包含一个RING基序，它作为DNA结合蛋白和核酸酶的已知酶功能是可消耗的。我已经证明这个结构域可以作为泛素连接酶，并提出它起调节作用。这一建议将允许实验室研究RAG1 n端结构域与通过双杂交筛选鉴定的几种蛋白质之间的相互作用。将研究几种特性，包括修饰状态、诱导亚细胞定位的变化、蛋白质翻转以及与这些蛋白质的相互作用是否影响重组靶标的选择。我们的目标是鉴定和表征涉及RAG1 N端结构域的蛋白质相互作用。预计这些基因将调控免疫系统发育的关键步骤，并可能导致与该过程相关的基因组不稳定。

DESCRIPTION (provided by applicant): The adaptive immune system of vertebrates depends upon a DNA recombination system, called V(D)J recombination, to generate the repertoire of immunoglobulin and T- cell receptors. This essential step also leads to chromosomal translocation when misapplied. Much of the regulation of this recombination process occurs at the initial stage when the DNA is cut. The proteins RAG1 and RAG2 form the site-specific nuclease that cuts the appropriate DNA targets and, it is believed, coordinate the recombination reaction. The RAG1 protein possesses a large N-terminal domain, containing a RING motif, which is expendable with respect to the known enzymatic functions as a DNA binding protein and a nuclease. I have shown that this domain can act as an ubiquitin ligase and propose that it plays a regulatory role. This proposal will allow the laboratory to study the interaction between the RAG1 N-terminal domain and several proteins that have been identified through a two-hybrid screen. Several properties will be examined including modification status, induced changes in subcellular localization, protein turn-over and whether the interaction with these proteins influences the choice of recombination targets. Our goal is to identify and characterize protein interactions involving the RAG1 N- terminal domain. These are expected to regulate a critical step in the development of the immune system, and may contribute to genomic instability associated with that process.