An interaction between a specified surface of the C-terminal domain of RecA protein and double-stranded DNA for homologous pairing.
RecA protein and its homologs catalyze homologous pairing of dsDNA and ssDNA, a critical reaction in homologous genetic recombination in various organisms from a virus, microbes to higher eukaryotes. In this reaction, RecA protein forms a nucleoprotein filament on ssDNA, which in turn binds to naked dsDNA for homology search. We suggested that the C-terminal domain of RecA protein plays a role in capturing the dsDNA. Here, we isolated the C-terminal domain as a soluble form and determined the solution structure by NMR spectroscopy. The overall folding of the NMR structure agrees with that of the corresponding part of the reported crystal structure, but a remarkable difference was found in a solvent-exposed region due to intermolecular contacts in the crystal. Then, we studied the interaction between the C-terminal domain and DNA, and found that significant chemical shift changes were induced in a specific region by titration with dsDNA. SsDNA induced a much smaller chemical shift perturbation. The difference of DNA concentrations to give the half-saturation of the chemical shift change showed a higher affinity of the C-terminal region toward dsDNA. Combined with our previous results, these provide direct evidence that the defined region in the C-terminal domain furnishes a binding surface for DNA.
Aihara
H
H
The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Japan.
Ito
Y
Y
Kurumizaka
H
H
Terada
T
T
Yokoyama
S
S
Shibata
T
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011995:Recombination, Genetic; D016415:Sequence Alignment
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D004247:DNA; D011938:Rec A Recombinases