Direct observation of DNA threading in flap endonuclease complexes
Faizah A AlMalki, Claudia S Flemming, Jing Zhang, Min Feng, Svetlana E Sedelnikova, Tom Ceska, John B Rafferty, Jon R Sayers & Peter J Artymiuk
Affiliations Contributions Corresponding authors
Nature Structural & Molecular Biology (2016) doi:10.1038/nsmb.3241
Received 07 February 2016 Accepted 10 May 2016 Published online 06 June 2016
Abstract
Maintenance of genome integrity requires that branched nucleic acid molecules be 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, which is enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate, thereby 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 'fly-casting, thread, bend and barb' mechanism.
Subject terms: DNA synthesis Enzyme mechanisms X-ray crystallography
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