RESOLUTION OF IMMOBILE CHI-STRUCTURES BY THE FLP RECOMBINASE OF 2-MU-M PLASMID

FLP is a conservative site-specific recombinase that is encoded by the 2 mu m plasmid of the yeast, Saccharomyces cerevisiae. FLP is member of the integrase family of recombinases that mediate the recombination reaction through a Holliday intermediate. The FLP recognition target (FRT) sites lie within two 599 bp inverted repeats of the 2 mu m plasmid. The minimal target contains two inverted FLP binding sites (13 bp) that surround an 8 bp core region. FLP nicks the top and the bottom strands of the FRT site at the margins of the core and these nicks are thought to be the sites of strand exchange. Hence, recombination generates heteroduplex DNA in the core region. It is known that heterology between the core regions of two FRT sites inhibits their ability to engage in recombination. It is possible that two homologous cores are required to allow the junction of the Holliday intermediate to branch migrate through the core during resolution. If so, an immobile Holliday junction point should inhibit the recombination activity of FLP in the same manner as a heterology between the cores of two double-stranded FRT sites. In order to test this prediction, we generated synthetic Holliday structures specific for FLP that had the junction immobilised at representative points within the FRT core. We used either sequence heterologies or nicked strands in order to immobilise the junction. We found that immobilisation of a Holliday junction within the core region did not inhibit resolution of the Holliday structure by FLP Hence, homology is not required for the resolution of the Holliday intermediate but rather, for an earlier step in the reaction.