Bacteriophage lambda site-specific recombination proceeds with a defined order of strand exchanges.
Previous work has established that integration of the genome of bacteriophage lambda into the chromosome of its bacterial host proceeds via two independent strand exchanges, which make and then resolve a Holliday-structure intermediate. We find that a phosphorothioate substitution at the site of exchange in one strand of a recombination site depresses the yield of Holliday structures much more than a similar substitution in the other strand. Furthermore, we show that the Holliday structures that accumulate in unblocked reactions have all been made by recombination of one particular pair of strands. We conclude that there is a strong bias in the choice of strands that initiate crossing-over. Excision, the recombination reaction that excises the integrated prophage, exhibits the same bias as integration. This proves, at least at the level of strand exchange, that excision is not the simple reversal of integration. We have altered the relative orientation of parts of the phage attachment site, attP, to demonstrate that the strand-exchange bias is determined not by the local environment around the point of exchange in the core of attP but by more distant elements in its arms. This suggests that the order of the strand exchanges is dictated by an asymmetry in the way that the nucleosome-like structure that forms at attP brings the bacterial site, attB, into juxtaposition prior to strand exchange. Finally, we use the altered attP to show that homology between attP and attB is most critical when it is adjacent to the point of strand exchange.
Kitts
P A
PA
Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, MD 20892.
Nash
H A
HA
D001287:Attachment Sites, Microbiological; D010582:Bacteriophage lambda / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli; D008242:Lysogeny; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D004279:DNA, Viral