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- Kim, Seog K., et al.
(författare)
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ENHANCEMENT OF BINDING RATE OF RECA PROTEIN TO DNA BY CARCINOGENIC BENZO A PYRENE DERIVATIVES AND SELECTIVE CHANGE OF ADDUCT CONFORMATION
- 1993
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Ingår i: Carcinogenesis. - : Oxford University Press (OUP). - 0143-3334 .- 1460-2180. ; 14:2, s. 311-313
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Tidskriftsartikel (refereegranskat)abstract
- The association kinetics of RecA protein from Escherichia coli to DNA is strongly enhanced if even a minor fraction of DNA bases has been modified by a carcinogenic (+)-anti metabolite of benzo[a]pyrene (BPDE). The enhancement is much smaller with the less carcinogenic (-)-anti enantiomer of BPDE suggesting the possibility that the RecA protein binds selectively to the proto-oncogenic target. Most importantly, the binding of RecA to DNA modified with the latter enantiomer is found to give rise to a reorganization of this BPDE adduct from an intercalation site into a minor groove site. This indicates that the binding mechanism of RecA is via intercalation of some amino acid moiety, a discovery that could explain the approximately 50% contour length increase of the DNA within its fibrous complex with RecA.
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| 2. |
- Kim, Seog K., et al.
(författare)
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ROLE OF DNA INTERCALATORS IN THE BINDING OF RECA TO DOUBLE-STRANDED DNA
- 1993
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 268:20, s. 14799-14804
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Tidskriftsartikel (refereegranskat)abstract
- RecA protein can bind to double-stranded DNA even without the cofactor ATP if a DNA intercalator such as ethidium bromide is present (Thresher R. J., and Griffith, J. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5056-5060). We have studied the structure and association kinetics of the ethidium-promoted DNA-RecA complex in order to understand the role of this intercalator in the DNA-RecA association process, information that could provide insight about the binding mechanism of RecA to DNA. Both linear dichroism and fluorescence measurements show that ethidium remains intercalated between the DNA bases in the RecA-DNA complex in the absence of ATP. Even in the presence of the ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), ethidium bromide shows some stimulating effect on the binding of RecA to DNA. The results indicate that the destacking of DNA bases is an important limiting step in the association of RecA to DNA (DNA is stretched in the ATPgammaS-RecA-DNA complex). In the presence of ATPgammaS, however, ethidium was extruded from DNA upon the binding of RecA. This result suggests that the binding mechanism of RecA to DNA may involve intercalation of one or more amino acid residues of RecA between the DNA bases. Such an intercalation would also be consistent with the stretching of DNA and the observation that the DNA bases remain in a (virtually stacked) perpendicular geometry (Takahashi, M., Kubista, M., and Norden, B. (1991) Biochemie (Paris) 73, 219-226; Norden, B., Elvingson, C., Kubista, M., Sjoberg, B., Ryberg, H., Ryberg, M., Mortensen, K., and Takahashi, M. (1992b) J. Mol. Biol. 226, 1175-1191).
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| 6. |
- Wittung, Pernilla, 1968, et al.
(författare)
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INTERACTIONS BETWEEN DNA-MOLECULES BOUND TO RECA FILAMENT - EFFECTS OF BASE COMPLEMENTARITY
- 1994
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 269:8, s. 5799-5803
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Tidskriftsartikel (refereegranskat)abstract
- 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 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 acrylamide provide information about the DNA environment in the complexes 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 increased fluorescence anisotropy shows that the mobility of the BPDE fluorophore is restricted in the Reck 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 Reck 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 Beck filament. BPDE-modified poly(dA) forms a duplex with poly(dT), whereupon the fluorescence of BPDE is strongly decreased. Binding of Reck 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 Beck filament.
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