Nonidentical DNA-binding sites of endonuclease NaeI recognize different families of sequences flanking the recognition site.
NaeI endonuclease uses a two-site binding mechanism to cleave substrate DNA: reaction-rate studies imply that occupancy of the second DNA site causes an allosteric change in the protein that enables DNA cleavage at the first site [Conrad, M., & Topal, M. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9707-9711]. Measurements of relative binding affinities for 14-base-pair DNA fragments containing the NaeI recognition sequence GCCGGC and various flanking sequences showed that the two DNA-binding sites are not identical. G.C-rich flanking sequences were preferred by the activator binding site, whereas A.T-rich flanking sequences were preferred by the substrate binding site: GGGTGCCGGCAGGG was preferred 8-fold more by the activator site but 14-fold less by the substrate site than TTTCGCCGGCGTTT. Substitution of pyrimidine or 7-deazapurine for purine immediately 3' to GCCGGC reduced DNA affinity for only the activator site by up to 26-fold, implying that the activator DNA-binding site requires N-7 base contacts immediately flanking GCCGGC. The implications of nonidentical DNA-binding sites, one of which binds a specific DNA site to allosterically activate the other, are discussed.
Yang
C C
CC
UNC Lineberger Comprehensive Cancer Center, University of North Carolina Medical School, Chapel Hill 27599-7295.
Topal
M D
MD
D000494:Allosteric Regulation; D017104:Bacteriophage M13 / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008969:Molecular Sequence Data
D004279:DNA, Viral; C060630:endodeoxyribonuclease NaeI; D015252:Deoxyribonucleases, Type II Site-Specific