Recognition of core-type DNA sites by lambda integrase.
Escherichia coli phage lambda integrase (Int) is a 40 kilodalton, 356 amino acid residue protein, which belongs to the lambda Int family of site-specific recombinases. The amino-terminal domain (residues 1 to 64) of Int binds to "arm-type" DNA sites, distant from the sites of DNA cleavage. The carboxy-terminal fragment, termed C65 (residues 65 to 356), binds "core-type" DNA sites and catalyzes cleavage and ligation at these sites. It has been further divided into two smaller domains, encompassing residues 65 to 169 and 170 to 356, respectively. The latter has been characterized and its crystal structure has been determined. Although this domain catalyzes the cleavage and rejoining of DNA strands it, unexpectedly, does not form electrophorectically stable complexes with core-type DNA. Here we have investigated the critical features of lambda Int binding to core-type DNA sites; especially, the role of the central 65 to 169 domain. To eliminate the complexities arising from lambda Int's heterobivalency we studied Int C65, which was shown to be as competent as Int, in binding to, and cleaving, core-type sites. Zero-length UV crosslinking was used to show that Ala125 and Ala126 make close contact with bases in the core-type DNA. Modification by pyridoxal 5'-phosphate was used to identify Lys103 at the protein-DNA interface. Since both of the identified loci are in the central domain, it was cloned and purified and found to bind to core-type DNA autonomously and specifically. The synergistic roles of the catalytic and the central, or core-binding (CB), domains in the interaction with core-type DNA are discussed for (Int and related DNA recombinases.
Tirumalai
R S
RS
Department of Biology and Medicine, Brown University Providence, RI 02912, USA.
Kwon
H J
HJ
Cardente
E H
EH
Ellenberger
T
T
Landy
A
A
D010582:Bacteriophage lambda / Q000201:enzymology*; D001665:Binding Sites / Q000235:genetics; D003090:Coliphages / Q000201:enzymology*; D003432:Cross-Linking Reagents / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry; D019426:Integrases / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011732:Pyridoxal Phosphate / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; D004247:DNA; D019426:Integrases; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D008239:Lysine