Swapping DNA strands and sensing homology without branch migration in lambda site-specific recombination.
BACKGROUND
Many site-specific recombinases act by forming and resolving branched Holliday junction intermediates. Previous findings have been consistent with models involving branch migration across the 'overlap region' of obligate homology, located between the staggered sites where the two single-strand exchanges occur. We have investigated the validity of such models in the case of bacteriophage lambda site-specific recombination.


RESULTS
By using synthetic lambda att-site Holliday junctions, incorporating sequence heterologies that impose constraints on branch migration, we have found that the optimal position of the junction for either top-strand or bottom-strand resolution by lambda integrase (Int) is not at the ends, but close to the middle of the seven base-pair overlap region. A minor shift of the branch point around the central base pair caused a remarkable switch in resolution bias. Our findings suggest that branch migration is limited to the central one to three base pairs of the overlap region. They lead to a new model for lambda site-specific recombination, in which there are two symmetrical swaps of two to three nucleotides each, linked by a central isomerization step that causes a change of the stacking interactions between the four junction arms. On the basis of isolated strand-joining reactions carried out by Int in the presence or absence of base complementarity, we propose that sequence homology is sensed during the annealing step prior to strand joining. The new model eliminates mechanistic complications associated with large helical rotations required by branch-migration models.


CONCLUSIONS
The results reported here suggest that the recognition of sequence homology in Int-dependent site-specific recombination does not rely primarily on branch migration. The property of cleaving Holliday junctions a few base pairs away from the crossover puts lambda Int into the same category as endonucleases that cleave Holliday junctions in homologous recombination.
Nunes-Düby
S E
SE
Department of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
Azaro
M A
MA
Landy
A
A
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics*; D019426:Integrases; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid
D004279:DNA, Viral; D004254:DNA Nucleotidyltransferases; D019426:Integrases