Purification and properties of Int-h, a variant protein involved in site-specific recombination of bacteriophage lambda.
Under physiological conditions, integration of lambda DNA into the Escherichia coli chromosome requires the direct participation of only two proteins, the viral int gene product and E. coli integration host factor (IHF). A variant of the int gene has been isolated that permits integrative recombination in cells mutant for one of the two subunits of IHF (Miller, H.I., Mozola, M.A., and Friedman, D.I. (1980) Cell 20, 721-729). In the present work, we have purified Int-h, the product of this variant gene. In contrast to the wild-type int gene product (Int+), which produces almost no recombinants in the absence of IHF, purified Int-h protein sponsors reduced but significant levels of integrative recombination in the absence of any E. coli supplement. This shows that the int gene encodes all the information necessary for the elementary steps in recombination and implies that IHF functions as an accessory protein. When supplemented by IHF, recombination promoted by Int-h resembles that promoted by Int+ in kinetics, stoichiometry of Int and IHF, and nature of the recombinant product. Under these conditions, Int-h uses supercoiled DNA more effectively than nonsupercoiled DNA as a substrate for recombination, as does Int+. However, in the absence of IHF, Int-h recombines supercoiled and nonsupercoiled substrates identically, indicating that IHF is an important part of the mechanism that senses the supercoiled state of the substrate DNA during recombination. A surprising difference in recombination carried out by Int-h in the presence or absence of IHF concerns the degree to which sites on the same circle recombine with one another as opposed to sites on sister molecules. In the presence of IHF, Int-h favors intramolecular recombination, as does Int+. However, in the absence of IHF, Int-h almost exclusively promotes intermolecular recombination.
D010582:Bacteriophage lambda / Q000235:genetics*; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005814:Genes, Viral; D014644:Genetic Variation; D019426:Integrases; D009154:Mutation; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification*
D004251:DNA Transposable Elements; D014764:Viral Proteins; D019426:Integrases; D004262:DNA Restriction Enzymes