2 research outputs found
Direct observation of DNA threading in flap endonuclease complexes
Maintenance of genome integrity requires that branched nucleic acid molecules are
accurately processed to produce double-helical DNA. Flap endonucleases are essential
enzymes that trim such branched molecules generated by Okazaki fragment synthesis during
replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in
complexes with intact DNA substrates, and products, at resolutions of 1.9–2.2 Å. They reveal
single-stranded DNA threading through a hole in the enzyme enclosed by an inverted Vshaped
helical arch straddling the active site. Residues lining the hole induce an unusual
barb-like conformation in the DNA substrate juxtaposing the scissile phosphate and essential
catalytic metal ions. A series of complexes and biochemical analyses show how the
substrate’s single-stranded branch approaches, threads through, and finally emerges on the far
side of the enzyme. Our studies suggest that substrate recognition involves an unusual “flycasting,
thread, bend and barb” mechanis