Protein-protein interactions directing resolvase site-specific recombination: a structure-function analysis.
Recombination catalyzed by the gamma delta resolvase requires assembly of a nucleo-protein complex, the synaptosome, whose structure is determined by resolvase-res and resolvase-resolvase interactions. In crystals of the resolvase catalytic domain, monomers of resolvase were closely associated with one another across three different dyad axes; one of these subunit contacts was shown to be an essential inter-dimer interaction. To investigate the relevance of the remaining two interfaces, we have made site-directed mutations at positions suggested by the structure. Cysteine substitutions were designed to link the interfaces covalently, mutations to arginine were used to disrupt intersubunit contacts, and mutations to tryptophan were used to study the hydrophobicity and solvent accessibility of potential interfaces by fluorescence quenching. Characterization of the mutant proteins has allowed us to identify the dimer interface of resolvase and to assign a structural role to a second intersubunit contact. The data presented here, together with our previous results, suggest that all three of the dyad-related intersubunit interactions observed in the crystal play specific roles in synapsis and recombination.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510.
R E
RE
Hughes
P A
PA
Rice
T A
TA
Steitz
N D
ND
Grindley
D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D003545:Cysteine / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004220:Disulfides / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D011995:Recombination, Genetic; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D019895:Transposases; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004220:Disulfides; D014364:Tryptophan; D001120:Arginine; D009713:Nucleotidyltransferases; D019895:Transposases; D003545:Cysteine