Mapping interactions between the catalytic domain of resolvase and its DNA substrate using cysteine-coupled EDTA-iron.
Single cysteine-substituted mutants of gamma delta resolvase have been covalently modified using a novel sulfhydryl-specific EDTA derivative, EDTA-2-aminoethyl 2-pyridyl disulfide (EPD). Iron, chelated by the coupled EDTA and in the presence of reducing agent, generates reactive oxygen species that result in localized cleavage of the DNA to which resolvase is bound. The procedure provides valuable information on two fronts. First, it allows the identification of regions or surfaces of the protein that are in close proximity to DNA even though they may not be part of the DNA-binding domain. Second, it allows identification of the portions of DNA that are closest to each EDTA-derivatized cysteine, since the DNA cleavages observed are highly localized and their efficiency drops rapidly as a function of the distance between the EDTA-Fe complex and the deoxyribose target. We have used the procedure to investigate the interaction of gamma delta resolvase with the three DNA binding sites that constitute its recombination substrate, res. The data indicate that the two N-terminal domains of a resolvase dimer interact symmetrically with site I, which contains the recombination cross-over point, but asymmetrically with the accessory sites, II and III. The patterns of DNA cleavage obtained with several different EDTA-coupled mutants have enabled us to propose a model for the interaction between resolvase and site I.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
J M
JM
Mazzarelli
M R
MR
Ermácora
R O
RO
Fox
N D
ND
Grindley
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004492:Edetic Acid / Q000378:metabolism; D006878:Hydroxides / Q000378:metabolism; D017665:Hydroxyl Radical; D007501:Iron / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019895:Transposases
D006878:Hydroxides; D017665:Hydroxyl Radical; D004247:DNA; D004492:Edetic Acid; D007501:Iron; D009713:Nucleotidyltransferases; D019895:Transposases; D003545:Cysteine