Studies Of Immunoglobulin Gene Rearrangement

我们继续研究V(D)J重组，基因片段的重排，用于组装完整的免疫球蛋白和T细胞受体编码区。这个过程是由RAG1和RAG2蛋白组成的重组酶启动的，它们共同作用，将双链断裂引入编码片段附近的DNA，并在此过程中将编码DNA的末端转化为共价连接的DNA发夹。因为类似的发夹是由一些相关的转座酶形成的，比如真核生物hAT转座酶的大群。我们正在跟进这些酶的类比，特别是因为它适用于使发夹成为可能所需的DNA结构的大扭曲。我们已经确定了两个色氨酸残基，它们可能参与结合从DNA中挤出的碱基，并且正在进一步测试这个模型。

We continue to study V(D)J recombination, the rearrangement of gene segments that is used to assemble intact immunoglobulin and T cell receptor coding regions. This process is initiated by a recombinase made up of the RAG1 and RAG2 proteins, which act together to introduce double-stranded breaks into the DNA adjacent to the coding segments, and in the process converts the ends of the coding DNA into covalently joined DNA hairpins. Because similar hairpins are formed by some related transposases, such as the large group of eukaryotic hAT transposases. we are following up the analogy with these enzymes, specifically as it applies to the large distortion of DNA structure required to make hairpinning possible. We have identified two tryptophan residues that may be involved in binding a base extruded from the DNA, and are working further to test this model. We are also investigating the binding of metal ions to RAG1. A tightly bound calcium ion appears to be necessary for activity, although calcium by itself does not support enzymatic activity, which requires magnesium or manganese. Other enzymes of this class operate by a two magnesium-ion mechanism, but in this case a third binding site may be involved. Work is also continuing on constructing variants of the RAG1 and RAG2 proteins, to be tested for their ability to crystallize in a specific complex with DNA. In collaboration with a group in the Netherlands, we are also testing for genetic transposition driven by RAG1/2 in mammalian cells. Previous work has shown that RAG1/2 has a transposase activity in vitro, but earlier attempts to find transposition in vivo have failed.