Abstract
We have subjected the FLP protein of the 2-micron plasmid to partial proteolysis by proteinase K and have found that FLP can be digested into two major proteinase K-resistant peptides of 21 and 13 kDa, respectively. The 21-kDa peptide contains a site-specific DNA-binding domain that binds to the FLP recognition target (FRT) site with an affinity similar to that observed for the native FLP protein. This peptide can induce DNA bending upon binding to a DNA fragment containing the FRT site, but the angle of the bend (approximately 24 degrees) is smaller in magnitude than that induced by the native FLP protein (60 degrees). The additional DNA bending induced by the interaction between two native FLP molecules bound to the FRT site is not observed with the 21-kDa DNA-binding peptide. Amino-terminal sequencing has been used to map this peptide to an internal region of FLP that begins at residue Leu-148. It is likely that the DNA-binding peptide includes the catalytic site of the FLP protein.
Highlights
From the Department of Medical Genetics, Medical Sciences Building, Uniuersity of Toronto, Toronto, Ontario M5S lA8, Canada
The 21-kDa peptide contains a site-specific DNA-binding domain that binds to the FLP recognition target (FRT) site with an affinity similar to that observed for the native FLP protein
This peptide can induce DNA bending upon binding to a DNA fragment containing the FRT site, but the angle of the bend is smaller in magnitude than that induced by the native FLP protein (60’)
Summary
Peptide includes the catalytic siteof the FLP protein. Proteolytic Digestion of FLP Generates a DNA-bindingPeptide-Previous attempts to use deletion analysis to identify the DNA-binding domainof F L P were unsuccessful, possibly. We have found that thespecificity of this isolated DNA-binding domain may be less stringent than thaot f native FLP (Fig. 6 A ) . we have not observed a DNase I footprint produced by the 21-kDa peptide,:' it should be pointed outthat even the binding of intact FLP toa single 13-bp symmetry element of the FRT site(formation of complex I) does not give a prominent DNase I footprint (Andrews et al, 1987). The FLP- orP21-induced FLP has two regions that are highly conserved among six bend is indicatedby the fact that theprotein-DNA complex migrates FLP proteins from six 2-pm-like plasmids of various yeast more slowly when the bending site (i.e.the FRT sitei)s in themiddle strains (Utatsuet aZ., 1987) They cover amino acid residues of the fragment ( M )than when it is at the end ( E ) .The bending 185-203 and 295-313 of 2-pm plasmid FLP. We have found that several mutations in these two conserved regions give a binding-deficient and bending-deficient phenotype: Three residues (His-305, Arg-308, and Tyr-343) are absolutely conserved among the integrase family of recominases (e.g. Int, Cre, andFLP) (Argos et al, 1986).It hasbeen shown that Tyr-343 is directly involved in DNA cleavage, Arg-308 may help this step and His-305 is essential for the strand exchange and religation
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