Spectroscopic observation of renaturation between polynucleotides with RecA in the presence of ATP hydrolysis.
To obtain mechanistic insights about RecA-promoted base pairing between complementary polynucleotides, the complex formation of RecA with poly(dA) and poly(dT) in the presence of ATP (and ATP-regenerating system) has been studied. The reaction was followed using a fluorescent probe, benzopyrenediolepoxide (BPDE), covalently attached to less than 1% of the adenine bases of poly(dA). BPDE is sensitive to its environment and has been found useful for detection of interactions between DNA strands, in the three binding positions of the RecA filament, in the presence of adenosine 5'-O-3-thiotriphosphate (ATP[S]) [Wittung, P., Nordén, B. & Takahashi, M. (1994) J. Biol. Chem. 269, 5799-5803]. The emission spectrum of RecA:BPDE-poly(dA) formed in the presence of ATP is similar to that observed with ATP[S] supporting similar structures of the complexes. However, the fluorescence anisotropy is considerably reduced, suggesting a higher degree of freedom of DNA in the presence of ATP hydrolysis. Upon addition of a complementary strand, poly(dT), to a preformed filament of RecA:BPDE-poly(dA) in the presence of ATP, the fluorescence intensity slowly decreases and a change of emission profile consistent with Watson-Crick base pairing is observed. This contrasts with the case of ATP[S] in which normal base pairing is never observed. Hence, ATP hydrolysis appears necessary for the RecA filament to be able to promote true renaturation. The renaturation reaction is found more effective when one of the complementary DNA strands is bound in the primary RecA DNA-binding position and the other is added as the third strand, but the reaction can also occur between DNA strands in any combination of binding positions in the RecA filament. This observation suggests the importance of the third DNA-binding position of the RecA filament. Renaturation between DNA strands in the other two combinations of binding positions is speculated to have a role in aborting the strand-exchange reaction when the strands are insufficiently complementary.
Wittung
P
P
Department of Physical Chemistry, Chalmers University of Technology, Gothenburg, Sweden.
Nordén
B
B
Takahashi
M
M
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001482:Base Composition; D004268:DNA-Binding Proteins; D005454:Fluorescence Polarization; D006868:Hydrolysis; D009695:Nucleic Acid Renaturation; D011061:Poly A / Q000378:metabolism*; D011071:Poly T / Q000378:metabolism*; D011938:Rec A Recombinases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D004268:DNA-Binding Proteins; D011061:Poly A; D011071:Poly T; C015465:poly(dA); D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases