Direct observation of DNA threading in flap endonuclease complexes.
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.
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK.
Faizah A
FA
AlMalki
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK.
Claudia S
CS
Flemming
Department of Infection, Immunity &Cardiovascular Disease, University of Sheffield, Sheffield, UK.
Jing
J
Zhang
Department of Infection, Immunity &Cardiovascular Disease, University of Sheffield, Sheffield, UK.
Min
M
Feng
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK.
Svetlana E
SE
Sedelnikova
UCB Pharma, Slough, UK.
Tom
T
Ceska
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK.|Krebs Institute, University of Sheffield, Sheffield, UK.
John B
JB
Rafferty
Department of Infection, Immunity &Cardiovascular Disease, University of Sheffield, Sheffield, UK.|Krebs Institute, University of Sheffield, Sheffield, UK.|Florey Institute, University of Sheffield, Sheffield, UK.
Jon R
0000-0002-5082-1443
JR
Sayers
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK.
Peter J
PJ
Artymiuk
D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006860:Hydrogen Bonding; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017906:Siphoviridae / Q000737:chemistry* / Q000201:enzymology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009841:Oligonucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; D005090:Exodeoxyribonucleases; C079044:T5 D15 exonuclease