The efficiency of mispaired ligations by lambda integrase is extremely sensitive to context.
The integrase protein (Int) of phage lambda is a well-studied representative of the tyrosine recombinase family, whose defining features are two sequential pairs of DNA cleavage/ligation reactions that proceed via a 3' phosphotyrosine covalent intermediate to first form and then resolve a Holliday junction recombination intermediate. We devised an assay that takes advantage of DNA hairpin formation at one Int target site to trap Int cleavages at a different target site, and thereby reveal iterative cycles of cleavage and ligation that would otherwise be undetected. Using this assay and others to compare wild-type Int and a mutant (R169D) defective in forming proper dimer/tetramer interfaces, we found that the efficiency of "bottom-strand" DNA cleavage by wild-type Int, but not R169D, is very sensitive to the base-pair at the "top-strand" cleavage site, seven base-pairs away. We show that this is related to the finding that hairpin formation involving ligation of a mispaired base is much faster for R169D than for wild-type Int, but only in the context of a multimeric complex. During resolution of Holliday junction recombination intermediates, wild-type Int, but not R169D, is very sensitive to homology at the sites of ligation. A long-sought insight from these results is that during Holliday junction resolution the tetrameric Int complex remains intact until after ligation of the product helices has been completed. This contrasts with models in which the second pair of DNA cleavages is a trigger for dissolution of the recombination complex.
Lee
Sang Yeol
SY
Division of Biology and Medicine, Brown University, Box G-J360, Providence, RI 02912, USA.
Landy
Arthur
A
D010582:Bacteriophage lambda / Q000201:enzymology / Q000235:genetics*; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D004254:DNA Nucleotidyltransferases; D019426:Integrases