An Assessment of the Z-DNA Forming Potential of Alternating dA-dT Stretches in Supercoiled Plasmids

The ability of a stretch of alternating dA-dT to adopt the left-handed Z form has been assessed by examining the behavior of the sequence d(CG)₆(TA)₄(CG)₆ contained in the plasmid pBR322. The structural transition occurring within this sequence as a function of negative superhelicity was analyzed by several methods, including (1) the supercoiling-dependent unwinding of the insert as determined by two-dimensional gel electrophoresis, (2) the binding of anti-Z-DNA antibodies to the insert, (3) the sensitivity of the sequence to a single strand specific endonuclease, and (4) the sensitivity of the insert to digestion by a restriction endonuclease that cuts within the d(CG)₆ segments when in the right-handed form. These studies have shown that in negatively supercoiled DNA the two d(CG)₆ portions of the insert adopt the Z form, while the central d(TA)₄ segment forms an underwound structure with a helical repeat that is best approximated as being intermediate between the B form and the Z form. A statistical mechanical treatment of the unwinding of the insert as a function of negative superhelicity provides an estimate of the minimum free energy required to convert an A-T bp from the B form to the Z form, as well as the free energy associated with the conversion of an A-T bp from the B form to the unwound form. These results strongly indicate that Z DNA is an unfavored structural alternative for stretches of d(AT)_n in negatively supercoiled DNA.