CA/TG sequence at the 5' end of oligo(A)-tracts strongly modulates DNA curvature.
An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5' end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAAC-CCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5' and 3' ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing of the sequences with AC/GT and CA/TG doublet junctions gives a similar cutting pattern in the A-tracts, which is quite different from that in the C-tracts, indicating that the oligo(A)-tracts have similar structures in the two oligomers. KMnO4 probing shows that the oligomer with a CA/TG doublet junction forms a kink that is responsible for its inherent curvature and unusual electrophoretic mobility. UV melting shows a reduced thermal stability of the duplex with CA/TG doublet junction, and circular dichroism (CD) studies indicate that a premelting transition occurs in the oligomer with CA/TG doublet step before global melting but not in the oligomer with AC/GT doublet step, which may correspond to thermally induced unbending of the oligomer. These observations indicate that the CA/TG doublet junction at the 5' end of the oligo(A)-tract has a crucial role in modulating the overall curvature in DNA.
Nagaich
A K
AK
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
Bhattacharyya
D
D
Brahmachari
S K
SK
Bansal
M
M
D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D017665:Hydroxyl Radical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011061:Poly A / Q000737:chemistry*; D011196:Potassium Permanganate; D013056:Spectrophotometry, Ultraviolet
D011196:Potassium Permanganate; D011061:Poly A; D017665:Hydroxyl Radical; D004247:DNA