Characterization of a mutation of bacteriophage lambda integrase. Putative role in core binding and strand exchange for a conserved residue.
Site-specific recombination is involved in processes ranging from resolution of bacterial chromosome dimers to adeno-associated viral integration and is a versatile tool for mammalian genetics. The bacteriophage lambda-encoded site-specific recombinase integrase (Int) is one of the best studied site-specific recombinases and mediates recombination via four distinct pathways. We have characterized a mutant version of lambda Int, IntT236I; this mutant can perform the bent-L pathway only, whereas the corresponding IntT236A mutant can perform bent-L, excision and integration pathways. Experiments with both IntT236I and IntT236A show that the hydroxyl group of threonine is necessary for wild-type recombination. Substitution of the threonine by serine leads to nearly complete rescue of the mutant phenotypes. In addition, our data show that the IntT236I mutant is defective partially due to obstructive steric interactions. Comparisons of crystal structures reveal that the threonine at residue 236 may play an important role in stabilizing recombination intermediates through solvent-mediated protein-DNA interactions at the core-binding sites and that the hydroxyl group is important for effective cleavage and Holliday junction formation. Our data also indicate that Int contacts the core sites differently in intermediates assembled in excisive versus bent-L recombination.
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Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, California 92182-4614, USA.
Segall
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D010582:Bacteriophage lambda / Q000201:enzymology*; D003198:Computer Simulation; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D019426:Integrases / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013912:Threonine / Q000378:metabolism
D013912:Threonine; D019426:Integrases