N-terminal 33 amino acid residues of Escherichia coli RecA protein contribute to its self-assembly.

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.