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
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
SUBSCRIPTION OR PAYMENT NEEDED/REQUER ASSINATURA OU PAGAMENTO: