title: N-terminal 33 amino acid residues of Escherichia coli RecA protein contribute to its self-assembly.
abstract: 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.
authors.affiliation: Department of Biology, Faculty of Science, Osaka University, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Mikawa
authors.forename: R
authors.initials: R
authors.lastname: Masui
authors.forename: T
authors.initials: T
authors.lastname: Ogawa
authors.forename: H
authors.initials: H
authors.lastname: Ogawa
authors.forename: S
authors.initials: S
authors.lastname: Kuramitsu
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D015870:Gene Expression; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011938:Rec A Recombinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D014357:Trypsin; D000251:Adenosine Triphosphatases; D008274:Magnesium