5hmm

Crystal Structure of T5 D15 Protein Co-crystallized with Metal IonsCrystal Structure of T5 D15 Protein Co-crystallized with Metal Ions

Structural highlights

5hmm is a 2 chain structure with sequence from Escherichia virus T5. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 1.5Å
Ligands:	, ,
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

FEN_BPT5 Catalyzes both the 5'-exonucleolytic and structure-specific endonucleolytic hydrolysis of DNA branched nucleic acid molecules and probably plays a role in viral genome replication (PubMed:9874768, PubMed:15077103, PubMed:10364212). Active on flap (branched duplex DNA containing a free single-stranded 5'-end), 5'overhangs and pseudo-Y structures (PubMed:9874768, PubMed:15077103, PubMed:10364212). The substrates require a free, single-stranded 5' end, with endonucleolytic hydrolysis occurring at the junction of double- and single-stranded DNA (PubMed:9874768). This function may be used for example to trim such branched molecules generated by Okazaki fragments synthesis during replication.[HAMAP-Rule:MF_04140]^[1] ^[2] ^[3]

Publication Abstract from PubMed

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 A. 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.

Direct observation of DNA threading in flap endonuclease complexes.,AlMalki FA, Flemming CS, Zhang J, Feng M, Sedelnikova SE, Ceska T, Rafferty JB, Sayers JR, Artymiuk PJ Nat Struct Mol Biol. 2016 Jun 6. doi: 10.1038/nsmb.3241. PMID:27273516^[4]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Pickering TJ, Garforth S, Sayers JR, Grasby JA. Variation in the steady state kinetic parameters of wild type and mutant T5 5'-3'-exonuclease with pH. Protonation of Lys-83 is critical for DNA binding. J Biol Chem. 1999 Jun 18;274(25):17711-7. PMID:10364212
↑ Feng M, Patel D, Dervan JJ, Ceska T, Suck D, Haq I, Sayers JR. Roles of divalent metal ions in flap endonuclease-substrate interactions. Nat Struct Mol Biol. 2004 May;11(5):450-6. Epub 2004 Apr 11. PMID:15077103 doi:10.1038/nsmb754
↑ Garforth SJ, Ceska TA, Suck D, Sayers JR. Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage. Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):38-43. PMID:9874768
↑ AlMalki FA, Flemming CS, Zhang J, Feng M, Sedelnikova SE, Ceska T, Rafferty JB, Sayers JR, Artymiuk PJ. Direct observation of DNA threading in flap endonuclease complexes. Nat Struct Mol Biol. 2016 Jun 6. doi: 10.1038/nsmb.3241. PMID:27273516 doi:http://dx.doi.org/10.1038/nsmb.3241

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OCA

[1] Pickering TJ, Garforth S, Sayers JR, Grasby JA. Variation in the steady state kinetic parameters of wild type and mutant T5 5'-3'-exonuclease with pH. Protonation of Lys-83 is critical for DNA binding. J Biol Chem. 1999 Jun 18;274(25):17711-7. PMID:10364212

[2] Feng M, Patel D, Dervan JJ, Ceska T, Suck D, Haq I, Sayers JR. Roles of divalent metal ions in flap endonuclease-substrate interactions. Nat Struct Mol Biol. 2004 May;11(5):450-6. Epub 2004 Apr 11. PMID:15077103 doi:10.1038/nsmb754

[3] Garforth SJ, Ceska TA, Suck D, Sayers JR. Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage. Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):38-43. PMID:9874768

[4] AlMalki FA, Flemming CS, Zhang J, Feng M, Sedelnikova SE, Ceska T, Rafferty JB, Sayers JR, Artymiuk PJ. Direct observation of DNA threading in flap endonuclease complexes. Nat Struct Mol Biol. 2016 Jun 6. doi: 10.1038/nsmb.3241. PMID:27273516 doi:http://dx.doi.org/10.1038/nsmb.3241

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