Resolution of tethered antiparallel and parallel holliday junctions by the Flp site-specific recombinase.
Members of the integrase family site-specific recombinases (also called the tyrosine family) bring about recombination in two steps by exchanging pairs of single strands at a time. The product of the first exchange reaction is a four-way DNA junction, the Holliday intermediate. The conformational dynamics by which the recombination complex "isomerizes" from the Holliday-forming to the Holliday-resolving mode are not well understood. Experiments with the lambda Int and Escherichia coli XerC/XerD systems imply that the strand configurations at the branch point of the protein-free junction dictate the resolution mode in the protein-bound junction. We have examined the question of strand bias during resolution for the Flp system by using a series of synthetic Holliday junctions that are conformationally constrained by local sequences or by strand tethering. We have not observed a strong resolution bias in favor of the strands designed to assume the "crossed" configuration within the unbound junction. The resolution patterns with antiparallel junctions in a variety of substrate contexts reveal either parity in strand choice, or only modest disparity. On the other hand, the highly biased resolutions observed in the case of tethered parallel junctions can be explained by the non-equivalence in protein occupancy of the DNA arms of these substrates and/or inefficient conversion of cleavage events to recombinants at the tethered ends.
Lee
J
J
Faculty of Applied Marine Sciences, Cheju University, Cheju City, 690756, South Korea.
Tribble
G
G
Jayaram
M
M
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018983:DNA Footprinting; D019175:DNA Methylation; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008957:Models, Genetic; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic / Q000235:genetics*; D013379:Substrate Specificity; D013463:Sulfuric Acid Esters / Q000378:metabolism
D004268:DNA-Binding Proteins; D013463:Sulfuric Acid Esters; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; C007482:dimethyl sulfate