Interactions between DNA molecules bound to RecA filament. Effects of base complementarity.
To gain information about the mechanism of RecA-promoted strand exchange reactions in genetic recombination, we have investigated the environment of and interactions between DNA strands accommodated in complexes with RecA protein. DNA bound in different sites in the RecA filament was tested by adding stoichiometric amounts of DNAs of variable base compositions. For this purpose, poly(dA) labeled by fluorescent benzo(a)pyrenediol epoxide (BPDE), which binds covalently to N-6 of adenine, was used. The fluorescence intensity, anisotropy, and quenching by acrylamide provide information about the DNA environment in the complexes with RecA. In the absence of extra DNA, binding of RecA to BPDE-poly(dA) only slightly affects both the intensity of the BPDE fluorescence and the accessibility of BPDE to acrylamide. However, a strongly increased fluorescence anisotropy shows that the mobility of the BPDE fluorophore is restricted in the RecA complex. Binding of a second and third single-stranded DNA to the RecA.BPDE-poly(dA) complex reduces the fluorescence intensity in a manner that depends on base complementarity. The change is large when the second and third DNAs are poly(dT), i.e. complementary to the already bound poly(dA) strand. In such a complex, the accessibility of BPDE to quencher is also reduced. When BPDE-poly(dA) was added as the second or third DNA in the RecA filament, the fluorescence intensity became smaller when the earlier bound DNA was complementary to the added DNA. These results indicate that all three DNA strands are interacting with each other in the RecA filament. BPDE-modified poly(dA) forms a duplex with poly(dT), whereupon the fluorescence of BPDE is strongly decreased. Binding of RecA to this duplex DNA increases the BPDE fluorescence, suggesting a destabilization of the duplex. Finally, we also find indications of base complementary interactions between single-stranded and double-stranded DNAs bound to the RecA filament.
Wittung
P
P
Department of Physical Chemistry, Chalmers University of Technology, Gothenburg, Sweden.
Nordén
B
B
Kim
S K
SK
Takahashi
M
M
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts; D005454:Fluorescence Polarization; D011061:Poly A / Q000378:metabolism; D011071:Poly T / Q000378:metabolism; D011938:Rec A Recombinases / Q000378:metabolism*
D018736:DNA Adducts; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D011061:Poly A; D011071:Poly T; C015465:poly(dA); D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA; D011938:Rec A Recombinases