Characterization of the oligomeric states of RecA protein: monomeric RecA protein can form a nucleoprotein filament.
Self-assembly of RecA protein in solution and on single-stranded DNA exerts a significant effect on the catalytic activities of this protein. To manipulate the self-association reaction, we examined the effects of various salts on the self-association of RecA from Thermus thermophilus (ttRecA) by circular dichroism spectroscopy and gel-filtration analysis. We showed that the self-association of ttRecA strongly depends on the kind and concentration of the salt, as well as on the protein concentration. Chaotropic ions were especially useful for obtaining RecA in its hexameric and monomeric states. On the basis of these observations, we were able to regulate the oligomeric states of ttRecA and we then examined the activity of RecA in various oligomeric states. Monomeric ttRecA bound to ssDNA and formed a nucleoprotein filament, which showed ssDNA-dependent ATPase activity. These results suggest that the monomeric form of RecA is an intermediate in filament formation on ssDNA.
Masui
R
R
Department of Biology, Graduate School of Science, Osaka University, Japan.
Mikawa
T
T
Kato
R
R
Kuramitsu
S
S
D002850:Chromatography, Gel; D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli; D009698:Nucleoproteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D011189:Potassium Chloride; D011485:Protein Binding; D011938:Rec A Recombinases / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D012492:Salts; D014508:Urea
D004277:DNA, Single-Stranded; D009698:Nucleoproteins; D012492:Salts; D011189:Potassium Chloride; D014508:Urea; D011938:Rec A Recombinases