Secondary structure of RecA in solution. The effects of cofactor, DNA and ionic conditions.
The interactions of RecA with double-stranded DNA and with cofactor adenosine 5'-[3-thio]triphosphate (ATP[S] an analog of ATP) have been characterized by circular dichroism (CD) spectroscopy in a search for conformational changes associated with the formation of helical RecA . ATP . DNA fibers. Upon interaction with the RecA protein the cofactor is found to be structurally perturbed, possibly towards the syn ribose form of ATP[S], while the secondary structure of RecA remains unaffected. By contrast, when the ATP[S] . RecA . DNA complex is formed, a distinct change of the protein CD spectrum near 200 nm is observed as a result of interaction of RecA with DNA. The main change occurs upon the binding of the first DNA molecule [RecA can bind up to three DNA molecules simultaneously; Takahashi, M., Kubista, M. & Nordén, B. (1991) Biochimie (Paris) 73, 219-226] and the effect appears to be independent of DNA sequence, suggesting a general change of protein conformation upon DNA binding. The CD of DNA is changed, indicating an alteration of the DNA structure, possibly related to stretching and unwinding. A small, reversible, decrease in the CD amplitude of RecA was observed when raising the temperature from 4 degrees C to 30 degrees C. The CD of RecA increases slightly with pH (up to 7.8) but is constant between pH 6.0 and 6.8. At pH below 6.0 or higher temperature (40 degrees C) slow irreversible denaturation of RecA occurs. The CD signal is effectively independent of salt, even in 2.2 M NaCl or 1 M sodium acetate, which is relevant regarding reported ATPase and coprotease activities promoted by salt. For high concentrations of magnesium (10 mM) at 30 degrees C the CD of RecA changes markedly and the appearance of light scattering indicates aggregation.
Wittung
P
P
Department of Physical Chemistry, Chalmers University of Technology, Gothenburg, Sweden.
Nordén
B
B
Takahashi
M
M
D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium; D009994:Osmolar Concentration; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011938:Rec A Recombinases / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature
D004247:DNA; D011938:Rec A Recombinases; D008274:Magnesium