The tyrosine-6 hydroxyl of gamma delta resolvase is not required for the DNA cleavage and rejoining reactions.
Site-specific recombinases of the resolvase and DNA invertase family all contain a tyrosine residue close to the N-terminus, and four residues away from a serine that has been implicated in catalysis of DNA strand breakage and reunion. To examine the role of this tyrosine in recombination, we have constructed a mutant of gamma delta resolvase in which the tyrosine (residue 6) is replaced by phenylalanine. Characterization of the Y6F mutant protein in vitro indicated that although it was highly defective in recombination, it could cleave DNA at the cross-over site, form a covalent resolvase-DNA complex and rejoin the cleaved cross-over site (usually restoring the parental site). These data rule out a direct role of the Tyr-6 hydroxyl as the nucleophile in the DNA cleavage reaction and strengthen the conclusion that this nucleophile is the nearby invariant serine residue, Ser-10. We conclude that Tyr-6 is essential for fully co-ordinated strand cleavage and exchange, but is dispensable for individual strand cleavage and religation reactions.
Leschziner
A E
AE
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
Boocock
M R
MR
Grindley
N D
ND
D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019426:Integrases; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D017354:Point Mutation / Q000235:genetics; D045522:Recombinases; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D019895:Transposases; D014443:Tyrosine / Q000737:chemistry*
D004268:DNA-Binding Proteins; D045522:Recombinases; D014443:Tyrosine; D004247:DNA; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D009713:Nucleotidyltransferases; D019895:Transposases; C115006:integron integrase IntI1; D004264:DNA Topoisomerases, Type I