Enhanced reactivity of a B-Z junction for cleavage by the restriction enzyme MboI.
We have been investigating the structure, dynamics, and ligand-binding properties of the interface that exists between a right-handed conformation and a left-handed conformation (i.e., a B-Z junction) in synthetic DNA oligomers. Since exo- and endonuclease activity is known to be sensitive to the conformation of the template DNA, we have designed and synthesized a DNA oligonucleotide of 20 base pairs (designated as BZ-III) with an MboI recognition site (GATC) at the location of a potential B-Z junction. The activity of the MboI enzyme toward this molecule and DNA oligomers that contain multiple MboI sites located at B-Z junctions was monitored in the absence and presence of the Z-conformation-inducing reagent cobalt hexaammine. In all cases, the activity of the enzyme was enhanced in the presence of cobalt hexaammine. The activity of MboI toward BZ-III, in the presence and absence of cobalt hexaammine, was also examined when the DNA oligomer is also in the presence of the DNA binding drugs actinomycin D, ametantrone, or ethidium bromide. In all cases, the activity of the enzyme was inhibited in the presence of drug. The results suggest that B-Z junctions are structurally unique and that this uniqueness may alter nuclease activity at sites in or near the junction.
Department of Chemistry, Florida International University, Miami 33199.
S A
SA
Winkle
M C
MC
Aloyo
N
N
Morales
T Y
TY
Zambrano
R D
RD
Sheardy
D001483:Base Sequence; D001665:Binding Sites; D003035:Cobalt / Q000494:pharmacology; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004996:Ethidium / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008942:Mitoxantrone / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects
D046911:Macromolecular Substances; C032300:hexaamminecobalt(II); D003609:Dactinomycin; D003035:Cobalt; D004247:DNA; D008942:Mitoxantrone; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases; D004996:Ethidium; C020182:ametantrone