Analysis of strand exchange and DNA binding of enhancer-independent Gin recombinase mutants.
The Gin recombination system of phage Mu mediates inversion of the DNA sequence between two sites (gix). In addition to Gin protein and gix sites, recombination requires an enhancer bound by the host factor FIS. We analyzed mutants of Gin that function in the absence of the enhancer and FIS and mediate deletion and intermolecular fusion in addition to inversion. The linking number changes caused by inversion imply that mutant Gin alone can form the same synaptic complex and can use the same strand exchange mechanism as the complete wild-type system. However, the linking number changes also reveal that unlike wild-type Gin, mutant Gin can recombine through more than one synaptic complex and can relax DNA in the absence of synapsis. This expanded repertoire allows mutant Gin to mediate DNA rearrangements not performed by wild-type Gin. Because mutant Gin, but not wild-type Gin, unwinds gix site DNA upon binding, we postulate that FIS and the enhancer function with (-) supercoiling to promote this unwinding with wild-type Gin. The analysis of the topological changes during DNA fusion shows that both the parallel gix site configuration and the right-handed rotation of the sites during exchange of wild-type Gin are a result of the (-) supercoiling of the substrate and the number of entrapped supercoils in the synaptic complex.
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
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Klippel
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Kanaar
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Kahmann
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Cozzarelli
D010583:Bacteriophage mu / Q000378:metabolism; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D009154:Mutation; D009690:Nucleic Acid Conformation; D010641:Phenotype; D011995:Recombination, Genetic
D004279:DNA, Viral; D004254:DNA Nucleotidyltransferases; C071957:Gin recombinase; D004264:DNA Topoisomerases, Type I