Regulation and Molecular Mechanisms of Antigen Receptor Gene Rearrangements

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 h RAG1 and RAG2 proteins in the presence of DNA bending proteins H1.Using magnetic beads，we have purified the SE complex and analyzed the SE DNA by DNaseI footprinting.It was found that the 7 mer rather than the 9 mer plays a primary role in the SE complex formation。It was also found that the 3‘-OH of the 7 mer 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.体外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 I D2κD 2 gene is responsible for the negative regulation of V I D2κD 2 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 I D2κD2-J I D2κD2 joining.Thus，the PU.1 motif alone appears to regulate both temporal and tissue-specific rearrangements of the Ig I D2κD 2 gene。

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.