Dynamics and structures of DNA: Long‐range effects of a 16 base‐pair (CG)8 sequence on secondary structure

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Abstract
The effects of inserting 16 base pair (bp) of alternating CG[(CG)8] near the middle of a much longer restriction fragment (1097 bp) are investigated by measuring various properties that are sensitive to secondary and tertiary structure. Results for this fragment are compared with those for a control fragment (1089 bp) with the identical sequence except at the insert. Another fragment (1382 bp), which contains a 296‐bp extension at the 5′‐end of the 1089‐bp control fragment, is also used as a secondary control in some experiments. When the 1097‐bp (CG)8 insert fragment is compared with the control fragments in 0.1M NaCl buffer, the (CG)8 insert is found to induce disproportionately large relative changes in the molar ellipticity at 273 nm ([θ)273], the torsion constant (α) measured by fluorescence polarization anisotropy, the optical melting profile, and the susceptibility to S1 nuclease. Estimates of the minimum distance over which the (CG)8 insert alters the secondary structure range from 330 to 550 bp. With increasing NaCl concentration, the 1097‐bp insert fragment undergoes a structural transition between 2.0 and 2.5M that is manifested in the apparent diffusion coefficient (Dplat) from dynamic light scattering at large scattering vector. This transition, which is not exhibited by the control DNAs, is presumed to involve formation of Z‐helix at the insert. However, the observed decrease in (Dplat) is attributed to an increase in bending rigidity, which perforce must be globally distributed far beyond the (CG)8 insert per se. In 4.25M NaCl (but not in 0.1M NaCl), the addition of 1 ethidium dye per 300 bp induces an extensive structural transition in the 1097 bp (CG)8 insert fragment. This transition, which also is not exhibited by the control DNAs, significantly decreases the bending rigidity, doubles [θ]273, and takes place on a time scale of a few days. Removal of ethidium and salt by dialysis vs 0.1M NaCl buffer restores the original properties of the 1097‐bp (CG)8 insert fragment. The present results are consistent with a (fluctuating, long‐range) description of the secondary structure in which a given short sequence transiently fluctuates among two or more distinct secondary structures that extend over much larger domains of variable position and size, and whose relative stabilities depend on distant as well as close‐lying base pairs. © 1993 John Wiley & Sons, Inc.