Characterization of the bacteriophage lambda excisionase (Xis) protein: the C-terminus is required for Xis-integrase cooperativity but not for DNA binding.
We have performed a mutational analysis of the xis gene of bacteriophage lambda. The Xis protein is 72 amino acids in length and required for excisive recombination. Twenty-six mutants of Xis were isolated that were impaired or deficient in lambda excision. Mutant proteins that contained amino acid substitutions in the N-terminal 49 amino acids of Xis were defective in excisive recombination and were unable to bind DNA. In contrast, one mutant protein containing a leucine to proline substitution at position 60 and two truncated proteins containing either the N-terminal 53 or 64 amino acids continued to bind lambda DNA, interact cooperatively with FIS and promote excision. However, these three mutants were unable to bind DNA cooperatively with Int. Cooperativity between wild-type Xis and Int required the presence of FIS, but not the Int core-type binding sites. This study shows that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision.
Numrych
T E
TE
Department of Microbiology, College of Medicine, University of Illinois, Urbana 61801.
Gumport
R I
RI
Gardner
J F
JF
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D019426:Integrases; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014764:Viral Proteins; D015678:Viral Structural Proteins / Q000235:genetics
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C063911:excisionase