The catalytic domain of lambda site-specific recombinase.
The Escherichia coli phage lambda integrase protein (Int) belongs to the large Int family of site-specific recombinases. It is a heterobivalent DNA binding protein that makes use of a high energy covalent phosphotyrosine intermediate to catalyze integrative and excisive recombination at specific chromosomal sites (att sites). A 293-amino acid carboxy-terminal fragment of Int (C65) has been cloned, characterized, and used to further dissect the protein. From this we have cloned and characterized a 188-amino acid, protease-resistant, carboxy-terminal fragment (C170) that we believe is the minimal catalytically competent domain of Int. C170 has topoisomerase activity and converts att suicide substrates to the covalent phosphotyrosine complexes characteristic of recombination intermediates. However, it does not show efficient binding to att site DNA in a native gel shift assay. We propose that lambda Int consists of three functional and structural domains: residues 1-64 specify recognition of "arm-type" DNA sequences distant from the region of strand exchange; residues 65-169 contribute to specific recognition of "core-type" sequences at the sites of strand exchange and possibly to protein-protein interactions; and residues 170-356 carry out the chemistry of DNA cleavage and ligation. The finding that the active site nucleophile Tyr-342 is in a uniquely protease-sensitive region complements and reinforces the recently solved C170 crystal structure, which places Tyr-342 at the center of a 17-amino acid flexible loop. It is proposed that C170 is likely to represent a generic Int family domain that thus affords a specific route to studying the chemistry of DNA cleavage and ligation in these recombinases.
Tirumalai
R S
RS
Division of Biology and Medicine, Brown University, Providence, RI 02912, USA.
Healey
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E
Landy
A
A
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism*; D010450:Endopeptidases; D004926:Escherichia coli / Q000821:virology; D019426:Integrases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D045522:Recombinases; D014443:Tyrosine
D010446:Peptide Fragments; D011994:Recombinant Proteins; D045522:Recombinases; D014443:Tyrosine; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C115006:integron integrase IntI1; D010450:Endopeptidases