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Studies of DNA dumbbells. II. Construction and characterization of DNA dumbbells with a 16 base-pair duplex stem and Tn end loops (n = 2, 3, 4, 6, 8, 10, 14)

Author:
Amaratunga, M., Snowden-Ifft, E., Wemmer, D.E., Benight, A.S.
Source:
Biopolymers 1992 v.32 no.7 pp. 865-879
ISSN:
0006-3525
Subject:
restriction mapping, heat stability, DNA, melting point, nucleotide sequences, DNA conformation, electrophoresis
Abstract:
The preparation and characterization of DNA dumbbells that contain the 16 base-pair duplex sequences (5')G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C(3') (set 1) and (5')G-C-A-T-C-A-T-C-G-A-T-G-A-T-G-C(3') (set 2) are reported. The dumbbells of set 1 have the duplex stem nucleated on both ends by T(n) (n = 2, 3, 4, 6, 8, 10, and 14) loops. The dumbbells of set 2 have T(n) (n = 2, 4, 8, 10) end loops. For the molecules of set 1, effects of end loop size on the electrophoretic mobility, CD and UV absorbance spectra, and cleavage by restriction enzymes, were investigated. Effects of loop size on the CD spectra and restriction enzyme cleavage of the molecules of set 2 were also examined. Optical melting curves of the molecules of set 1 were collected as a function of sodium ion concentration from 30 to 120 mM. These investigations revealed that as loop size decreases, the electrophoretic mobilities, rates of enzyme cleavage, and optical melting temperatures increase. For end loops with at least three T's the observed increases are inversely proportional to loop size. The behavior of the dumbbell with T(2) end loops departs from this linear dependence and is anomalous in every experimental context. For molecules with end loops comprised of at least four T's CD spectra were virtually indistinguishable. However, these spectra differed considerably from the CD spectrum of the T(2)-looped molecule. The CD spectrum of the dumbbell with T(3) end loops displayed features common to the dumbbells with larger loops and T(2) end loops. Thermodynamic evidence that the terminal G-C base pairs (bps) nucleating the T(2) end loops were intact was obtained from a comparison of the melting temperature of this molecule with that of a DNA dumbbell containing the 14 central bps of the set 1 duplex sequence linked instead by end loops comprised of the four base sequence, C-T-T-C. The t(m) of this latter molecule was determined to be 9 degrees C less than that of the former dumbbell assumed to contain a 16-bp stem and T(2) end loops. One- and two-dimensional proton nmr measurements performed on the dumbbell of set 2 with T(2) end loops and the same sequence with unpaired T's on the ends also verified formation of the T(2) end loops, indicated the duplex residues of the dumbbell were in the B form, and showed that the first 3 bps adjoining the loops are distorted. Results are compared with several recently published relevant studies.
Agid:
1383713