The gamma delta resolvase bends the res site into a recombinogenic complex.
We have characterized complexes between the gamma delta resolvase and its recombination site, res, using both a gel retardation assay and DNase I cleavage. The mobility of resolvase-res complexes in polyacrylamide gels is sensitive to the location of res within the DNA fragment and is at a minimum when res is at its center. This behavior is characteristic of a protein-dependent bend. By the same assay we have found that bends are induced upon the binding of resolvase to each of the three individual binding sites that constitute res. In the wild-type res, the centers of binding sites I and II are 53 bp apart and the central section of the intersite DNA is sensitive to DNase I cleavage. We find that insertions of 10 or 21 bp (one or two turns of the DNA helix) have no discernible effect on the ability of res to recombine or to form complexes with resolvase. However, insertions of short segment (e.g. 6 or 17 bp) equivalent to nonintegral numbers of helical turns, inhibit recombination and prevent the formation of the normally compact resolvase-res complex. Complexes of resolvase with res containing 10 or 21 bp insertions exhibit a pattern of enhanced and suppressed DNase I cleavages that suggest that the intersite segment is curved. This curvature requires both that site I and II are appropriately spaced, and that site III is also present and occupied.
Salvo
J J
JJ
Department of Molecular Biophysics and Biochemistry, Yale University Medical School, New Haven, CT 06510.
Grindley
N D
ND
D001665:Binding Sites; D004247:DNA / Q000032:analysis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D009690:Nucleic Acid Conformation; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D015183:Restriction Mapping; D019895:Transposases
D004247:DNA; D009713:Nucleotidyltransferases; D019895:Transposases