Functional structures of the recA protein found by chimera analysis.
We developed a novel genetic method for finding functional regions of a protein by the analysis of chimeras formed between homologous proteins. Sets of chimeric genes were made by intramolecular homologous recombination in a linearized plasmid DNA carrying both recA genes of Escherichia coli and Pseudomonas aeruginosa. A recBCsbcA strain of E. coli was used for isolation of plasmids carrying recombinants between these genes. Examination of properties of E. coli strains deleting the recA gene and carrying a plasmid with a chimeric gene shows that chimera formation at certain positions inactivates a RecA function. Frequently, all chimeras with a junction in a certain region of the protein inactivate a function. Rather than a direct effect of the presence of the junction at a particular position, mismatching of the regions both sides of the junction that are derived from the different species is responsible for the inactivation. For a chimeric protein to be functional, certain pairs of sequences in different regions of the protein must derive from the same parent. Four pairs of such sequences were found: two are involved in activities for genetic recombination and for resistance to ultraviolet light irradiation and the others in formation of active oligomers. Regions defined by these sequences are located in the looped regions of the protein. A pair of regions may co-operate to form a functional folded structure.
Ogawa
T
T
Department of Biology, Faculty of Science, Osaka University, Japan.
Shinohara
A
A
Ogawa
H
H
Tomizawa
J
J
D000595:Amino Acid Sequence; D002678:Chimera; D004307:Dose-Response Relationship, Radiation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014466:Ultraviolet Rays
D011993:Recombinant Fusion Proteins; D011938:Rec A Recombinases