GCN4 eukaryotic transcription factor/FokI endonuclease-mediated 'Achilles' heel cleavage': quantitative study of protein-DNA interaction.
Three proteins, yeast transcription regulatory protein GCN4, M.FokI DNA methyltransferase and R.FokI restriction endonuclease (ENase) were used to attain specific cleavage of DNA at the 18-20-bp GCN4 recognition site. This is a novel version of the 'Achilles' heel cleavage' (AC) technique [Koob et al., Science 241 (1988) 1084-1086]. Since the method employs a class-IIS ENase (R.FokI), which cleaves the DNA outside of its recognition sequence, it leaves the overlapping GCN4-binding intact. Thus, the same GCN4 site can be used in consecutive cleavage reactions. This novel GCN4-IIS-AC technique was applied to study the protein-DNA interaction. Quantitative analysis of the effect of temperature, reaction time, and GCN4 and M.FokI concentrations allowed determination of the GCN4-DNA complex half-life, which was found to be 7 h at 30 degrees C, 18 h at 22 degrees C and over 24 h at 10 degrees C. In addition, conditions for controlled, partial GCN4-IIS-AC digestion of DNA were determined, and applied to the physical mapping of large genomes.
Skowron
P M
PM
University of Wisconsin-Madison, McArdle Laboratory for Cancer Research, Madison, WI 53706, USA.
Harasimowicz
R
R
Rutkowska
S M
SM
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004274:DNA, Recombinant / Q000378:metabolism; D004268:DNA-Binding Proteins; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D016521:Electrophoresis, Gel, Pulsed-Field; D005656:Fungal Proteins / Q000378:metabolism*; D006207:Half-Life; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C058668:DNA modification methylase FokI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D011494:Protein Kinases; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific