B-Z junctions in supercoiled pRW751 DNA contain unpaired bases or non-Watson-Crick base pairs.
Structural distortions on the boundary between right-handed and left-handed DNA segments in negatively supercoiled plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of osmium tetroxide, pyridine and glyoxal. These two probes react preferentially with single-stranded DNA, but only the latter requires non-paired bases for the reaction. Nuclease S1 and testing of the inhibition of BamHI cleavage (whose recognition sequences GGATCC lie on the "outer" boundaries between the (dC-dG)n and the pBR322 nucleotide sequence) were used to detect the site-specific chemical modification in pRW751. As a result of glyoxal treatment BamHI cleavage was strongly inhibited in topoisomeric samples whose superhelical density was sufficiently negative to stabilize the (dC-dG)n segments in the left-handed form. Osmium tetroxide, pyridine modification resulted in a similar inhibition of BamHI cleavage and in a formation of nuclease S1 sensitive sites. The results suggest that the "outer" B-Z junctions in pRW751 contain one or few non-paired bases or non-Watson-Crick base pairs.
Palecek
E
E
Institute of Biophysics, Czechoslovak Academy of Sciences, Brno.
Boubliková
P
P
Nejedlý
K
K
Galazka
G
G
Klysik
J
J
D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000648:ultrastructure*; D004274:DNA, Recombinant; D004278:DNA, Superhelical / Q000187:drug effects / Q000648:ultrastructure*; D015245:Deoxyribonuclease BamHI / Q000037:antagonists & inhibitors; D006037:Glyoxal / Q000494:pharmacology; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009993:Osmium Tetroxide / Q000494:pharmacology; D010957:Plasmids; D011725:Pyridines / Q000494:pharmacology; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D004274:DNA, Recombinant; D004278:DNA, Superhelical; D011725:Pyridines; D006037:Glyoxal; D004247:DNA; D015245:Deoxyribonuclease BamHI; D015719:Single-Strand Specific DNA and RNA Endonucleases; C023666:pyridine; D009993:Osmium Tetroxide