The structural dynamics of mismatched base pairs in duplex DNA have been studied by time-resolved fluorescence anisotropy decay measurements on a series of duplex oligodeoxynucleotides of the general type d[CGG(AP)GGC].d[GCCXCCG], where AP is the fluorescent adenine analogue 2-aminopurine and X = T, A, G, or C. The anisotropy decay is caused by internal rotations of AP within the duplex, which occur on the picosecond time scale, and by overall rotational diffusion of the duplex. The correlation time and angular range of internal rotation of AP vary among the series of AP.X mismatches, showing that the native DNA bases differ in their ability to influence the motion of AP. These differences are correlated with the strength of base-pairing interactions in the various AP.X mismatches. The interactions are strongest with X = T or C. The ability to discern differences in the strength of base-pairing interactions at a specific site in DNA by observing their effect on the dynamics of base motion is a novel aspect of the present study. The extent of AP stacking within the duplex is also determined in this study since it influences the excited-state quenching of AP. AP is thus shown to be extrahelical in the AP.G mismatch. The association state of the AP-containing oligodeoxynucleotide strand is determined from the temperature-dependent tumbling correlation time. An oligodeoxynucleotide triplex is formed with a particular base sequence in a pH-dependent manner.
The interaction of a fluorescent duplex DNA oligomer with the Klenow fragment of DNA polymerase I from Escherichia coli has been studied in solution by using time-resolved fluorescence spectroscopy. An aminonaphthalenesulfonate (dansyl) fluorescent probe was linked by a propyl chain to a C5-modified uridine base located at a specific site in the primer strand of the DNA oligomer. The fluorescent oligomer bound tightly to the Klenow fragment (KD = 7.9 nM), and the probe's position within the DNA-protein complex was varied by stepwise elongation of the primer strand upon addition of the appropriate deoxynucleoside triphosphates. The decay of the total fluorescence intensity and the polarization anisotropy were measured with a picosecond laser and a time-correlated single photon counting system. The fluorescence lifetimes, the correlation time for internal rotation, and the angular range of internal rotation varied according to the probe's position within the DNA-protein complex. These results showed that five or six bases of the primer strand upstream of the 3' terminus were in contact with the protein and that within this contact region there were differences in the degree of solvent accessibility and the closeness of contact. Further, a minor binding mode of the DNA-protein complex was identified, on the basis of heterogeneity of the probe environment observed when the probe was positioned seven bases upstream from the primer 3' terminus, which resulted in a distinctive "dip and rise" in the anisotropy decay. Experiments with an epoxy-terminated DNA oligomer and a site-directed mutant protein established that the labeled DNA was binding at the polymerase active site (major form) and at the spatially distinct 3'----5' exonuclease active site (minor form). The abundance of each of these distinct binding modes of the DNA-protein complex was estimated under solution conditions by analyzing the anisotropy decay of the dansyl probe. About 12% of the labeled DNA was bound at the 3'----5' exonuclease site. This method should be useful for investigating the editing mechanism of this important enzyme.
The technique of long-range electronic energy transfer has been used to obtain structural data on nucleic acids in solution. Fluorescent chromophores have been covalently attached via an aminoethyl linker to the 5'-terminus of a synthetic oligonucleotide.Duplexes were formed with an energy donor, fluorescein, at one end and an acceptor, tetramethyl rhodamine, at the other. The rate of energy transfer has been determined from the fluorescence decay of the donor which was measured with a picosecond photon counting system. The effects of dye:DNA interactions cause additional quenching of the donor fluorescence and must be considered in the interpretation of the energy transfer experiments. An apparent donor:acceptor distance of 40.9 A was calculated for a 12 b.p. labelled duplex.
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