In vivo assay of protein-protein interactions in Hin-mediated DNA inversion.
In order to form the catalytic nucleoprotein complex called the invertasome in the Hin-mediated DNA inversion reaction, interactions of the DNA-binding proteins Hin and Fis are required. Assays for these protein-protein interactions have been exploited with protein cross-linkers in vitro. In this study, an in vivo assay system that probes protein-protein interactions was developed. The formation of a DNA loop generated by protein interactions resulted in transcriptional repression of an artificially designed operon, which in turn increased the chance of survival of Escherichia coli host cells in a streptomycin-containing medium. Using this system, we were able to assay the Hin-Hin interaction that results in the pairing of the two recombination sites and protein interactions that result in the formation of the invertasome. This assay system also led us to find that an individual Hin dimer bound on a recombination site can form a stable complex with Fis bound on the recombinational enhancer; this finding has never been observed in in vitro studies. Possible pathways toward the formation of the invertasome are discussed based on the assay results for a previously reported Hin mutant.
Lee
S Y
SY
Department of Biology, College of Natural Sciences, Chungnam National University, Taejon 305-764, Korea.
Lee
H J
HJ
Lee
H
H
Kim
S
S
Cho
E H
EH
Lim
H M
HM
D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics; D007446:Chromosome Inversion; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; D009154:Mutation
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; C440502:integration host factor, E coli; D004254:DNA Nucleotidyltransferases; C059997:Hin recombinase