A site-targeted recombinant nuclease probe of DNA structure.
A genetically engineered lac repressor/T7 endonuclease hybrid protein shows repressor-like binding toward restriction fragments carrying the lac operator. In addition, fragments carrying the operator near a particular pBR322 sequence are cleaved into specific products. Cleavage occurs at precise positions within that sequence, is independent of the orientation of the operator, is inhibited by isopropyl-1-thio-beta-D-galactopyranoside, and is observed when the target is separated from the operator by at least as few as 150 and as many as 240 base pairs. This evidence indicates that the hybrid protein is a site-directed nuclease that requires the following two structural elements for activity: the lac operator and a target. Repressor-like binding directs the enzyme to the operator and nearby single-stranded DNA targets. The discovery of an unusual target in a well-studied DNA sequence is evidence of the power of this approach for probing unusual structures in non-supercoiled duplex DNA.
Panayotatos
N
N
Michigan Biotechnology Institute, Lansing 48909.
Bãckman
S
S
D001667:Binding, Competitive; D015342:DNA Probes / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D043223:Deoxyribonuclease IV (Phage T4-Induced); D015252:Deoxyribonucleases, Type II Site-Specific; D004706:Endodeoxyribonucleases / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D010957:Plasmids; D012097:Repressor Proteins / Q000378:metabolism; D015183:Restriction Mapping / Q000379:methods; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics
D015342:DNA Probes; D004274:DNA, Recombinant; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C040158:GTAC-specific type II deoxyribonucleases