INTERACTIONS BETWEEN DNA-MOLECULES BOUND TO RECA FILAMENT - EFFECTS OF BASE COMPLEMENTARITY

To gain information about the mechanism of RecA-promoted strand exchan ge reactions in genetic recombination, we have investigated the enviro nment of and interactions between DNA strands accommodated in complexe s with Beck protein. DNA bound in different sites in the Reck 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 ac rylamide provide information about the DNA environment in the complexe s with Reck In the absence of extra DNA, binding of Reck to BPDE-poly( dA) only slightly affects both the intensity of the BPDE fluorescence and the accessibility of BPDE to acrylamide. However, a strongly incre ased fluorescence anisotropy shows that the mobility of the BPDE fluor ophore is restricted in the Reck complex. Binding of a second and thir d single-stranded DNA to the RecA.BPDE-poly(dA) complex reduces the fl uorescence 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-pol y(dA) was added as the second or third DNA in the Reck filament, the f luorescence intensity became smaller when the earlier bound DNA was co mplementary to the added DNA. These results indicate that all three DN A strands are interacting with each other in the Beck filament. BPDE-m odified poly(dA) forms a duplex with poly(dT), whereupon the fluoresce nce of BPDE is strongly decreased. Binding of Reck to this duplex DNA increases the BPDE fluorescence, suggesting a destabilization of the d uplex. Finally, we also find indications of base complementary interac tions between single-stranded and double-stranded DNAs bound to the Be ck filament.