Interaction of the NH2- and COOH-terminal domains of the FLP recombinase with the FLP recognition target sequence.
The FLP protein that is encoded by the 2-microns plasmid of yeast Saccharomyces cerevisiae is a 45-kDa site-specific recombinase that belongs to the Int family of recombination proteins. FLP catalyzes a recombination event within the plasmid by binding specifically to each of three 13-base pair (bp) symmetry elements of the FLP recognition target (FRT). We have shown previously that partial proteolysis of the FLP protein by proteinase K resulted in a COOH-terminal fragment of size 32 kDa (P32) and an NH2-terminal fragment of 13 kDa (P13). In this study we have used footprinting with dimethyl sulfate to show that P32 binds specifically to the outer 9 bp of the 13 bp symmetry element. Binding of P13 alone to the FRT site was not detectable in this assay. However, when P13 and P32 were incubated together with the FRT site, protection of the remaining 4-bp region of the symmetry element was observed. To confirm these results we used bromodeoxyuridine (BrdU)-dependent UV cross-linking. P32 became cross-linked to the substrate that contained BrdU substitutions in the outer 9 bp of a 13-bp symmetry element, but not to one with the BrdU substitutions in the inner 4 bp. Reciprocally P13 cross-linked to the latter substrate but not the former. Cross-linking was both BrdU and ultraviolet light-dependent. This study indicates that the COOH-terminal domain (P32) of FLP recognizes the outer 9 bp of the 13-bp symmetry element, whereas its NH2-terminal domain (P13) is needed for protection of the inner 4 bp of each symmetry element.
Panigrahi
G B
GB
Department of Molecular and Medical Genetics, University of Toronto, Canada.
Sadowski
P D
PD
D000225:Adenine / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001973:Bromodeoxyuridine; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D006147:Guanine / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000528:radiation effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D004271:DNA, Fungal; D005656:Fungal Proteins; D010455:Peptides; D006147:Guanine; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D003850:Deoxyribonuclease I; D001973:Bromodeoxyuridine; D000225:Adenine