Inhibition of restriction endonuclease cleavage due to site-specific chemical modification of the B-Z junction in supercoiled DNA.
Structural distortions on the boundary between right-handed B and left-handed Z DNA segments in plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of chemical probes. Samples of supercoiled DNA were treated with the respective chemical probe, linearized with EcoRI and inhibition of BamHI (whose recognition sequence GGATCC lies on the boundary between the (dC-dG)n segments and the pBR322 nucleotide sequence) cleavage was tested. Treatment with osmium tetroxide in the presence of pyridine or 2,2'-bipyridine, respectively, resulted in a strong inhibition of the BamHI cleavage at both restriction sites, provided the (dC-dG)n segments were in the left-handed form. In the presence of 2,2'-bipyridine submillimolar concentrations of OsO4 (at 26 degrees C) were sufficient to induce the inhibition of BamHI. Chloroacetaldehyde was used as a probe reacting selectively with atoms involved in the Watson-Crick hydrogen bonding. Similarly as in the case of osmium tetroxide treatment of pRW751 with this agent resulted in the inhibition of BamHI cleavage. It was concluded that the B-Z junction regions in pRW751 contain few solitary bases with disturbed hydrogen bonding or non-Watson-Crick base pairs.
Palecek
E
E
Institute of Biophysics, Czechoslovak Academy of Sciences, Brno.
Boublíková
P
P
Galazka
G
G
Klysik
J
J
D000079:Acetaldehyde / Q000031:analogs & derivatives; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004278:DNA, Superhelical; D004587:Electrophoresis, Agar Gel; D009690:Nucleic Acid Conformation; D010957:Plasmids
D004278:DNA, Superhelical; C004656:chloroacetaldehyde; D004262:DNA Restriction Enzymes; D000079:Acetaldehyde