Studies Of Immunoglobulin Gene Rearrangement

我们对V(D)J重组的研究在过去一年里一直在继续，V(D)J重组是用于组装完整的免疫球蛋白和T细胞受体编码区的基因片段的重组。这个过程是由RAG1和RAG2蛋白组成的双链重组酶启动的，这两个蛋白共同作用，将双链断裂引入编码片段附近的DNA中。在相同的反应中，它们将编码DNA的末端转化为共价连接的DNA发夹。类似的发夹是由一些相关的转座酶形成的，例如真核细胞中的一大组HAT转座酶。我们正在继续这个类比，试图解释DNA结构的巨大扭曲，这是使发夹成为可能所必需的。我们已经确定了两个色氨酸残基，它们可能参与与DNA挤出的碱基结合，从而使DNA更灵活。为了支持这一模型，我们发现，移除切割位点的关键碱基(以产生基本结构)允许在通常不会发生的条件下切割DNA，并且还可以挽救原本阻止切割的色氨酸突变。 构建RAG1和RAG2蛋白变体的工作也在继续，以测试它们在与DNA的特定复合体中结晶的能力。

Our research on V(D)J recombination, the rearrangement of gene segments that is used to assemble intact immunoglobulin and T cell receptor coding regions, has continued in the past year. This process is initiated by a bipartite 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. In the same raction they convert the ends of the coding DNA into covalently joined DNA hairpins. Similar hairpins are formed by some related transposases, such as the large group of eukaryotic hAT transposases. and we are following up this analogy to try to explain the large distortion of DNA structure that is necessary to make hairpinning possible. We have identified two tryptophan residues that may be involved in binding a base extruded from the DNA, thus making the DNA more flexible. In support of this model, we have found that removal of a critical base at the cleavage site ( to generate an abasic structure) allows cutting of the DNA under conditions where it would not normally occur, and can also rescue tryptophan mutations that otherwise block cleavage. 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.