Resolution of tethered antiparallel and parallel Holliday junctions by theFlp site-specific recombinase

Members of the integrase family site-specific recombinases (also called the tyrosine family) bring about recombination in two steps by exchanging pair s of single strands at a time. The product of the first exchange reaction i s a four-way DNA junction, the Holliday intermediate. The conformational dy namics by which the recombination complex "isomerizes" from the Holliday-fo rming to the Holliday-resolving mode are not well understood. Experiments w ith the lambda Int and Escherichia coli XerC/XerD systems imply that the st rand configurations at the branch point of the protein-free junction dictat e the resolution mode in the protein-bound junction. We have examined the q uestion of strand bias during resolution for the Flp system by using a seri es of synthetic Holliday junctions that are conformationally constrained by local sequences or by strand tethering. We have not observed a strong reso lution bias in favor of the strands designed to assume the "crossed" config uration within the unbound junction. The resolution patterns with antiparal lel junctions in a variety of substrate contexts reveal either parity in st rand choice, or only modest disparity. On the other hand, the highly biased resolutions observed in the case of tethered parallel junctions can be exp lained by the non-equivalence in protein occupancy of the DNA arms of these substrates and/or inefficient conversion of cleavage events to recombinant s at the tethered ends. (C) 2000 Academic Press.