Mikawa T, Masui R, Ogawa T, Ogawa H, Kuramitsu S
Department of Biology, Faculty of Science, Osaka University, Japan.
J Mol Biol. 1995 Jul 21;250(4):471-83. doi: 10.1006/jmbi.1995.0391.
To identify the functional domains in the RecA protein, we prepared the truncated RecA protein lacking its N-terminal 33 amino acid residues by limited tryptic digestion and found that this truncated protein was inefficient at self-assembly. To investigate the function of the N-terminal region further, we constructed the N-terminal truncated recA gene lacking the portion corresponding to the N-terminal 33 residues and prepared a large amount of its gene product. This truncated protein could bind to ATP, but it was defective in self-assembly, binding to single-stranded (ss)DNA and hydrolysis of ATP under normal conditions, although no significant alteration in its stability in comparison with the wild-type protein was observed. In the presence of MgCl2, however, this truncated protein could self-assemble, although a higher protein concentration and longer time than for the wild-type protein were required to complete the process. This truncated protein inhibited the ssDNA-dependent ATPase and ssDNA-binding activities of the wild-type protein. Furthermore, gel filtration chromatography showed that this truncated protein interacted with the wild-type protein and reduced the apparent size of its aggregates. These results suggest that this truncated protein interfered with polymerization of the wild-type protein via a direct protein-protein interaction, which resulted in inhibition of ssDNA-binding and ssDNA-dependent ATP hydrolysis. On the basis of these observations, we concluded that the N-terminal 33 amino acid residues of the RecA protein play an important role not only in protein-protein interaction but also in regulation of the self-assembly process.
为了鉴定RecA蛋白中的功能结构域，我们通过有限的胰蛋白酶消化制备了缺失其N端33个氨基酸残基的截短型RecA蛋白，发现这种截短型蛋白在自我组装方面效率低下。为了进一步研究N端区域的功能，我们构建了缺失对应于N端33个残基部分的N端截短型recA基因，并制备了大量其基因产物。这种截短型蛋白能够结合ATP，但在正常条件下，它在自我组装、与单链（ss）DNA结合以及ATP水解方面存在缺陷，尽管与野生型蛋白相比其稳定性没有明显改变。然而，在MgCl2存在的情况下，这种截短型蛋白能够自我组装，尽管完成该过程所需的蛋白浓度比野生型蛋白更高且时间更长。这种截短型蛋白抑制了野生型蛋白的ssDNA依赖性ATP酶活性和ssDNA结合活性。此外，凝胶过滤色谱显示这种截短型蛋白与野生型蛋白相互作用并减小了其聚集体的表观大小。这些结果表明，这种截短型蛋白通过直接的蛋白质-蛋白质相互作用干扰了野生型蛋白的聚合，从而导致ssDNA结合和ssDNA依赖性ATP水解受到抑制。基于这些观察结果，我们得出结论，RecA蛋白的N端33个氨基酸残基不仅在蛋白质-蛋白质相互作用中起重要作用，而且在自我组装过程调控中也起重要作用。
