Regulation and Molecular Mechanisms of Antigen Receptor Gene Rearrangements

我们已经研究了重组信号序列（RSSs）和重组激活基因（RAGs）之间的相互作用。与已发表的Hin模型确认了序列特定RSS-RAG交互作用的神经元结构域中RAG 1蛋白和9聚DNA中偶联主要和小凹槽之间的RAG-RSS足迹数据的比较。当两个RSSs在V（D）J连接中被清除时，信号端（SE）复合体由RAG 1和RAG 2蛋白在DNA弯曲蛋白HMG 1中形成。使用磁性珠子，我们已经净化了SE复合体，并通过DNaseI脚印分析了SE DNA。它发现了七个人比九个人在SE复杂形成中的主要角色。我们还发现了7个人中的3'-OH在12 - 23-RSS复合体中磷酸化的情况。3'-磷酸盐是从载体DNA或RNA转移的，由跨式esterization后转移的。体外分析证实了这种3'-磷酸化预防了SE复合体的转移。These results indicate that the 3'-phosphorylation may have an active role in preventing harm？transposition of an excision product of V（D）J joining。We have shown that the PU.1 binding motif （GAGGAA） in the 3'-enhancer of the Igy D2 gene is responsible for the negative regulation of Vy D2-J y D2 joining。在转录激活中，PU.1动机与PIP和BSAP动机合作地在一个积极的标记中，它们位于同一增强核心区域。有趣的是， base substitutions in either the PIP or the BSAP motif did not affect the B/T or pro-B/pre-B specificity of Vy D2-Jy D2 joining。Thus，PU.1动机单独出现对Igy-D2 κ y-D2基因的临时和专题-具体的叙述。

We have studied the interaction between recombination signal sequences (RSSs) and protein products of the recombination-activating genes (RAGs). Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin homeodomain of the RAG1 protein and adjacent major and minor grooves of the 9mer DNA.When two RSSs are cleaved in V(D)J joining, the signal-end (SE) complex is formed with RAG1 and RAG2 proteins in the presence of DNA bending protein HMG1. Using magnetic beads, we have purified the SE complex and analyzed the SE DNA by DNaseI footprinting. It was found that the 7mer rather than the 9mer plays a primary role in the SE complex formation. It was also found that the 3'-OH of the 7mer is phosphorylated in the SE complex in both 12- and 23-RSS's after the cleavage. The 3'-phosphate is transferred from carrier DNA or RNA by trans-esterification after nicking of the carrier. In vitro assays demonstrated that the 3'-phosphorylation prevents the transposition of the SE complex. These results indicate that the 3'-phosphorylation may have an active role in preventing harmful transposition of an excision product of V(D)J joining.We have shown that the PU.1 binding motif (GAGGAA) in the 3'-enhancer of the IgィイD2κィエD2 gene is responsible for the negative regulation of VィイD2κィエD2-JィイD2κィエD2 joining. In the transcriptional activation, the PU.1 motif acts in a positive manner cooperatively with the PIP and BSAP motifs, which are located in the same enhancer core region. Interestingly, base substitutions in either the PIP or the BSAP motif did not affect the B/T or pro-B/pre-B specificity of VィイD2κィエD2-JィイD2κィエD2 joining. Thus, the PU.1 motif alone appears to regulate both temporal and tissue-specific rearrangements of the IgィイD2κィエD2 gene.