Professor Jon Sayers
We usually think about our DNA as being double-helical in structure, but in fact, DNA adopts a number of different shapes as part of normal biological processes. For example, branched DNA molecules occur in all living organisms from bacteria to Homo sapiens. These branched DNA structures have to be trimmed precisely to restore the double helix. Flap endonucleases, 5′ nucleases or 5′-3′ exonucleases are some of the names given to a group of ubiquitous structure-specific nucleases that can cleave branched DNA. Some viruses even carry genes encoding their own FEN enzymes. Apart from being essential for all cells, flap endonuclease (FENs) are also widely used in biotechnology e.g. in genotyping, quantitative PCR, polymorphism screening and molecular biology. For example, bacteriophage T5 exonuclease (a FEN despite its name) was first cloned by Sayers and is now employed in the widely used Gibson cloning strategy. The Sayers lab studies FENs as well as other DNA binding proteins combining site directed mutagenesis, biochemistry and structural approaches to unravel the mechanisms and also to work towards engineering novel reagents.
- 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 Jul;23(7):640-6. doi: 10.1038/nsmb.3241. Epub 2016 Jun 6. PubMed PMID: 27273516; PubMed Central PMCID: PMC4939078.
- Anstey-Gilbert CS, Hemsworth GR, Flemming CS, Hodskinson MR, Zhang J, Sedelnikova SE, Stillman TJ, Sayers JR, Artymiuk PJ. The structure of Escherichia coli ExoIX–implications for DNA binding and catalysis in flap endonucleases. Nucleic Acids Res. 2013 Sep;41(17):8357-67. doi: 10.1093/nar/gkt591. Epub 2013 Jul 2. PubMed PMID: 23821668; PubMed Central PMCID: PMC3783174.
- Wong IN, Sayers JR, Sanders CM. Characterization of an unusual bipolar helicase encoded by bacteriophage T5. Nucleic Acids Res. 2013 Apr;41(8):4587-600. doi: 10.1093/nar/gkt105. Epub 2013 Feb 21. PubMed PMID: 23435232; PubMed Central PMCID: PMC3632103.
- Tomlinson CG, Syson K, Sengerová B, Atack JM, Sayers JR, Swanson L, Tainer JA, Williams NH, Grasby JA. Neutralizing mutations of carboxylates that bind metal 2 in T5 flap endonuclease result in an enzyme that still requires two metal ions. J Biol Chem. 2011 Sep 2;286(35):30878-87. doi: 10.1074/jbc.M111.230391. Epub 2011 Jul 6. PubMed PMID: 21734257; PubMed Central PMCID: PMC3162448.
- Allen LM, Hodskinson MR, Sayers JR. Active site substitutions delineate distinct classes of eubacterial flap endonuclease. Biochem J. 2009 Mar 1;418(2):285-92. doi: 10.1042/BJ20081637. PubMed PMID: 19000038; PubMed Central PMCID: PMC2650880.
- Hodskinson MR, Allen LM, Thomson DP, Sayers JR. Molecular interactions of Escherichia coli ExoIX and identification of its associated 3′-5′ exonuclease activity. Nucleic Acids Res. 2007;35(12):4094-102. Epub 2007 Jun 12. PubMed PMID: 17567612; PubMed Central PMCID: PMC1919509.
- 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. PubMed PMID: 15077103.
- Dervan JJ, Feng M, Patel D, Grasby JA, Artymiuk PJ, Ceska TA, Sayers JR. Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding. Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8542-7. PubMed PMID: 12084915; PubMed Central PMCID: PMC124304.
- 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. PubMed PMID: 9874768; PubMed Central PMCID: PMC15089.
- Ceska TA, Sayers JR, Stier G, Suck D. A helical arch allowing single-stranded DNA to thread through T5 5′-exonuclease. Nature. 1996 Jul 4;382(6586):90-3. PubMed PMID: 8657312.