| 1. |
- Hsu, D. S., et al.
(författare)
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FLOW LINEAR DICHROISM AND ELECTRON-MICROSCOPIC ANALYSIS OF PROTEIN-DNA COMPLEXES OF A MUTANT UVRB PROTEIN THAT BINDS TO BUT CANNOT KINK DNA
- 1994
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 241:5, s. 645-650
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Tidskriftsartikel (refereegranskat)abstract
- (A)BC excinuclease of Escherichia coli is the enzymatic activity resulting from sequential and partially overlapping actions of UvrA, UvrB, and UvrC protein. UvrA is a molecular matchmaker which promotes the formation of a stable UvrB-damaged DNA complex in which the DNA is kinked by about 130 degrees. The UvrB-DNA complex is then recognized by UvrC) and two incisions are made in the DNA by the joint actions of UvrC and UvrB. A mutant of UvrB (D478A) can be loaded onto the DNA but it does not interact with UvrC to cause a nick 3' to the lesion. Based on the lack of a DNase-I-hypersensitive site in the footprint of the mutant, it was proposed that the lack of incision was due to the inability of the mutant UvrB to kink the DNA. In the current study we have investigated the interaction of the mutant UvrB with DNA using two biophysical methods, flow linear dichroism and electron microscopy. Both methods reveal that the mutant UvrB is unable to bend DNA.
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| 2. |
- 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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