Mak C H, Strandtmann J, Wu L C
Department of Medical Biochemistry, Ohio State Biochemistry Program, Ohio State University, Columbus 43210.
Nucleic Acids Res. 1994 Feb 11;22(3):383-90. doi: 10.1093/nar/22.3.383.
The murine DNA binding protein Rc binds to the heptamer motif of the V(D)J recombination signal sequences and to the kappa B motif of the immunoglobulin enhancer. Bacterial fusion proteins for Rc and DNA ligands of Rc form multiple protein-DNA complexes in electrophoretic mobility shift assays (EMSA). Large complexes formation is favored by an increased Rc concentration. In order to determine the architecture of these complexes, the apparent molecular weights of the protein-DNA complexes were first determined by their gel mobilities. The data suggest that Rc binds to its DNA ligands as dimers, tetramers, and multiples of tetramers. The inference that Rc binds DNA as dimers was substantiated by the formation of chimeric complexes when two electrophoretically distinguishable Rc proteins were employed in EMSA. Methylation interference experiments show that there are no contiguous protein binding sites evident in the DNA of the larger complexes. Apparently, multimerization occurs via protein-protein interactions. Such interaction was demonstrated by the formation of Rc dimers and tetramers in a chemical crosslinking experiment. Significantly, the multimerization of DNA-bound Rc could be involved in bringing the variable region gene segments together for the somatic V(D)J recombination.
小鼠DNA结合蛋白Rc可与V(D)J重排信号序列的七聚体基序以及免疫球蛋白增强子的κB基序结合。在电泳迁移率变动分析（EMSA）中，Rc的细菌融合蛋白及其DNA配体形成多种蛋白质-DNA复合物。Rc浓度增加有利于形成大的复合物。为了确定这些复合物的结构，首先通过凝胶迁移率测定蛋白质-DNA复合物的表观分子量。数据表明，Rc以二聚体、四聚体以及四聚体的倍数形式与其DNA配体结合。当在EMSA中使用两种电泳可区分的Rc蛋白时，嵌合复合物的形成证实了Rc以二聚体形式结合DNA的推断。甲基化干扰实验表明，在较大复合物的DNA中没有明显相邻的蛋白质结合位点。显然，多聚化是通过蛋白质-蛋白质相互作用发生的。在化学交联实验中，Rc二聚体和四聚体的形成证明了这种相互作用。值得注意的是，与DNA结合的Rc的多聚化可能参与将可变区基因片段聚集在一起进行体细胞V(D)J重排。
