Analysis of subunit interaction by introducing disulfide bonds at the dimerization domain of Hin recombinase.
The Hin recombinase exists as a homodimer in solution and binds to its recombination site (hix) as a dimer (Glasgow, A. C., Bruist, M. F., and Simon M. I. (1989) J. Biol. Chem. 264, 10072-10082). Previous mutational and structural studies of related proteins suggested the location of a putative dimerization domain. In order to probe the function of this region of the protein, cysteine residues were introduced at each of the seven positions comprising the domain. Proteins containing cysteine substitutions at positions 101 and 104 were able to form disulfide bonds spontaneously in crude extracts. M101C showed wild type inversion activity only if the cysteine residue was not engaged in a disulfide bond. This mutant, which is cross-linked by a disulfide bond at the dimeric interface, was found to be defective in the DNA cleavage step during inversion. H107C displayed the wild type DNA cleavage activity, even though its DNA binding activity was not detected by three different binding assays. This suggests that it maintains specificity for DNA binding but dissociates rapidly after binding. The remaining inversion-defective mutants fell into two groups. One group lost its capacity to specifically bind to DNA (G102C, F105C, and F106C), and the other group (R103C and F104C), while able to bind to DNA, was defective in subsequent steps of the inversion reaction. Characteristics of these mutants led me to postulate that movement of subunits at the dimerization interface is critical during DNA binding and during formation of the invertasome, which is the recombination structure. Furthermore, the relative position of subunits within the dimer may be important for maintaining stable DNA binding.
Lim
H M
HM
Department of Biology, College of Natural Sciences, Chungnam National University, Taejon, Korea.
D000595:Amino Acid Sequence; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011108:Polymers; D017386:Sequence Homology, Amino Acid
D004268:DNA-Binding Proteins; D004220:Disulfides; D011108:Polymers; D004254:DNA Nucleotidyltransferases; C059997:Hin recombinase; D004229:Dithiothreitol