Site-Specific Chemical Modification of B-Z Junctions in Supercoiled DNA as Detected by Nuclease SI Digestion, Inhibition of Restriction Cleavage and Nucleotide Sequencing

Abstract

Structural distortions on the boundary between right-handed and left-handed segments in the superhelical plasmid pPK2 (a derivative of pUC 19 containing (dC-dG)_n segments cloned into polylinker) were studied by means of chemical probes. Strong osmium tetroxide, pyridine (Os, py) modification of DNA at native superhelical density (σ) was found in four thymines surrounding the (dC-dG)₁₃ segment. These results correlated with restriction cleavage inhibition (due to modification): BamHI cleavage was strongly inhibited, unlike the neighbouring XbaI and SalI (weak or no inhibition). In the (dC-dG)₈segment considerably weaker modification of the B-Z junctions was observed, accompanied by weak inhibition of BamHI cleavage, while the neighbouring Smal and KpnI were not affected. Os, py modification of DNA at native σ was not detected by nuclease SI cleavage at and (dC-dG)_n segment. However, this enzyme recognized and cleaved at the B-Z junction, osmium modified at more negative σ. The results obtained with the glyoxal and diethyl pyrocarbonate modification support the idea of very narrow B-Z junctions at native σ.