Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence.
The Flp protein catalyzes a site-specific recombination reaction between two 47 bp DNA sites without the assistance of any other protein or cofactor. The Flp recognition target (FRT) site consists of three nearly identical sequences, two of which are separated by an 8 bp spacer sequence. In order to gain insight into this remarkable protein-DNA interaction we used a variety of chemical probe methods and the missing nucleoside experiment to examine Flp binding. Hydroxyl radical footprints of Flp bound to a recombinationally-competent site fall on opposite faces of canonical B-DNA. The 8 bp spacer region between the two Flp binding sites becomes reactive towards 5-phenyl-1,10-phenanthroline.copper upon Flp binding, indicating that once bound by Flp, this segment of DNA is not in the B-form. Missing nucleoside analysis reveals that within each binding site the presence of two nucleosides on the top strand and four on the bottom, are required for formation of a fully-occupied FRT site. In contrast, loss of any nucleoside in the three binding sites in the FRT interferes with formation of lower-occupancy complexes. DNA molecules with gaps in the 8 bp spacer region are over-represented in complexes with either two or three binding sites occupied by Flp, evidence that DNA flexibility facilitates the cooperative interaction of Flp protomers bound to a recombinationally-active site.
Kimball
A S
AS
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
Kimball
M L
ML
Jayaram
M
M
Tullius
T D
TD
D001483:Base Sequence; D001665:Binding Sites; D003300:Copper; D019327:Copper Sulfate; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004492:Edetic Acid / Q000031:analogs & derivatives; D017665:Hydroxyl Radical; D007502:Iron Chelating Agents; D008969:Molecular Sequence Data; D009705:Nucleosides; D010618:Phenanthrolines; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic
D004268:DNA-Binding Proteins; D007502:Iron Chelating Agents; D009705:Nucleosides; D010618:Phenanthrolines; D011994:Recombinant Proteins; C038200:methidiumpropyl-EDTA-iron(II); D017665:Hydroxyl Radical; C090893:5-phenyl-1,10-phenanthroline; D003300:Copper; D004247:DNA; D004492:Edetic Acid; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019327:Copper Sulfate