Site-specific recombination by mutants of Tn21 resolvase with DNA recognition functions from Tn3 resolvase.
The resolvases from the transposons Tn3 and Tn21 are homologous proteins but they possess distinct specificities for the DNA sequence at their respective res sites. The DNA binding domain of resolvase contains an amino acid sequence that can be aligned with the helix-turn-helix motif of other DNA binding proteins. Mutations in the gene for Tn21 resolvase were made by replacing the section of DNA that codes for the helix-turn-helix with synthetic oligonucleotides. Each mutation substituted one amino acid in Tn21 resolvase with either the corresponding residue from Tn3 resolvase or a residue that lacks hydrogen bonding functions. The ability of these proteins to mediate recombination between res sites from either Tn21 or Tn3 was measured in vivo and in vitro. With one exception, where a glutamate residue had been replaced by leucine, the activity of these mutants was similar to that of wild-type Tn21 resolvase. A further mutation was made in which the complete recognition helix of Tn21 resolvase was replaced with that from Tn3 resolvase. This protein retained activity in recombining Tn21 res sites, though at a reduced level relative to wild-type; the reduction can be assigned entirely to weakened binding to this DNA. Neither this mutant nor any other derivative of Tn21 resolvase had any detectable activity for recombination between res sites from Tn3. The exchange of this section of amino acid sequence between the two resolvases is therefore insufficient to alter the DNA sequence specificity for recombination.
Ackroyd
A J
AJ
Department of Biochemistry, University of Bristol, U.K.
Avila
P
P
Parker
C N
CN
Halford
S E
SE
D000595:Amino Acid Sequence; D001483:Base Sequence; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic / Q000502:physiology*; D013329:Structure-Activity Relationship; D019895:Transposases; D045564:Transposon Resolvases
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D009713:Nucleotidyltransferases; C473490:Tn21 resolvase; D019895:Transposases; D045564:Transposon Resolvases