Cooperative binding of Tn3 resolvase monomers to a functionally asymmetric binding site.
BACKGROUND
The inverted repeat is a common feature of protein-binding sites in DNA. The two-fold symmetry of the inverted repeat corresponds to the two-fold symmetry of the protein that binds to it. In most natural inverted-repeat binding sites, however, the DNA sequence does not have perfect two-fold symmetry. Our study of how a site-specific recombinase recognizes an inverted-repeat binding site indicates that such sequence asymmetry can be functionally important.


RESULTS
Tn3 resolvase forms two complexes with the 34 base-pair binding site II of its recombination region, res. A resolvase monomer first binds at the left end of the site; a second monomer then binds cooperatively at the right end. In both complexes, the DNA is bent by resolvase. In contrast, the closely related gamma delta resolvase binds to site II mainly as a dimer. Insertion of 5 or 10 base pairs at the centre of the site does not prevent cooperative binding of two Tn3 resolvase subunits. The fully occupied site II has a very asymmetric structure. Reversal of the orientation of site II in res blocks recombination; thus, its asymmetric properties are functionally important. We propose a structure for the two-subunit complex formed with site II, based on our results and by analogy with the co-crystal structure of gamma delta resolvase bound to res site I.


CONCLUSIONS
Deviations from perfect inverted-repeat symmetry in a resolvase-binding site lead to ordered binding of subunits, structural asymmetry of resolvase-DNA complexes, and asymmetric function.
Biomedical Research Centre, Dundee University, Ninewells Hospital and Medical School, UK.
D G
DG
Blake
M R
MR
Boocock
D J
DJ
Sherratt
W M
WM
Stark
D001483:Base Sequence; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D019895:Transposases
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D004254:DNA Nucleotidyltransferases; D019895:Transposases