Binding of recA protein to Z-form DNA studied with circular and linear dichroism spectroscopy.
Binding of RecA to poly(dG-m5dC) and poly(dG-dC) under B- and Z-form conditions was studied using circular dichroism (CD) and linear dichroism (LD). LD revealed a quantitative binding of RecA to Mg2+-induced Z-form poly(dG-m5dC) with a stoichiometry of 3.1 base pairs/RecA monomer, which is slightly larger than the 2.7 base pairs observed for the B-form. The LD spectra indicate a preferentially perpendicular orientation of DNA bases and a rather parallel orientation of the tryptophan residues relative to the fiber axis in both complexes. The association rate of RecA to Z-form DNA was found to be slower than to B-form. CD measurements showed that the polynucleotide conformation is retained upon RecA binding, and CD and LD confirm that RecA binds to both forms of DNA. The Mg2+-induced Z-form is shown to be retransformed into B-form, both in free and in RecA-complexed polynucleotides by addition of NaCl, whereas the B----Z transition cannot be induced by addition of Mg2+ when the polynucleotide is complexed with RecA. From this it is inferred that RecA does not stabilize the Z-conformation of the polynucleotide but that it can kinetically "freeze" the polynucleotide in its B-conformation. On all essential points, the same conclusions were also reached in a corresponding study of unmethylated poly(dG-dC) with the Z-form induced by Mn2+.
Takahashi
M
M
Institut de Biologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Strasbourg, France.
Kubista
M
M
Nordén
B
B
D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D011089:Polydeoxyribonucleotides; C016681:poly(dG).poly(dC); C029988:poly(dG-m(5)dC).poly(dG-m(5)dC); D004247:DNA; D011938:Rec A Recombinases; D008274:Magnesium