Use of monoacetyl-4-hydroxyaminoquinoline 1-oxide to probe contacts between guanines and protein in the minor and major grooves of DNA. Interaction of Escherichia coli integration host factor with its recognition site in the early promoter and transposition enhancer of bacteriophage Mu.
Monoacetyl-4-hydroxyaminoquinoline 1-oxide (Ac-HAQO) reacts with DNA to form adducts at the C8- and N2-positions of guanine and with the N6-position of adenine. Only the N2-guanine adduct blocks the 3'-5' exonuclease action of phage T4 DNA polymerase. Piperidine treatment cleaves the DNA at sites bearing C8-guanine adducts. The N2-position of guanine lies in the minor groove of DNA, whereas the C8-position of guanine occupies the major groove. We have taken advantage of these characteristics to employ Ac-HAQO in conjunction with either T4 DNA polymerase or piperidine in a footprinting technique to probe the interaction of the Escherichia coli integration host factor (IHF) with its binding site. We show that when IHF binds to its recognition site both the N2- and C8-positions of guanines are protected from modification by AcHAQO. In addition, the binding of IHF to DNA was prevented when either an N2- or a C8-AQO adduct was present in the binding site. When dimethylsulfate was used as the footprinting reagent, IHF protected against methylation of the N3 position of adenine in the minor groove but not the N7 position of guanine in the major groove. The difference in results obtained with the two reagents is ascribed to their relative sizes. Both DMS and AcHAQO are excluded by IHF from the minor groove, but only the larger AcHAQO molecule is excluded from the major groove.
Panigrahi
G B
GB
Department of Biochemistry, University of Western Ontario, London, Canada.
Walker
I G
IG
D000634:Aminoquinolines; D010583:Bacteriophage mu / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry*; D004742:Enhancer Elements, Genetic; D006147:Guanine / Q000737:chemistry*; D034982:Integration Host Factors; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic
D000634:Aminoquinolines; D002352:Carrier Proteins; D004269:DNA, Bacterial; D034982:Integration Host Factors; D015335:Molecular Probes; D006147:Guanine; C032982:4-acetoxyaminoquinoline 1-oxide