Professor Jane A. Grasby
Professor of Biological Chemistry
Telephone: +44 (0) 114 222 9478
Professor Grasby obtained her BSc in Chemistry from the University of Birmingham in 1988. After obtaining her PhD in Biochemistry from the University of Southampton in 1992, she became a Research Fellow at the Laboratory of Molecular Biology in Cambridge. In 1994 she was appointed to a Lectureship at the University of Sheffield, where she was promoted to Senior Lecturer in 1999, Reader in 2002 and a Personal Chair in 2013. Prof. Grasby has acted as Chair of the Faculty of Science Equality Diversity Committee.
Catalysis, protein and nucleic structure and function, nucleic acid chemistry, molecular recognition, enzymology
Organic Chemistry; Biological Chemistry
The interactions and reactions of nucleic acids are fundamental to life. Our research has seeks to understand these processes using a range of techniques including chemical synthesis of modified nucleic acids, molecular biology, enzymology, biophysics (fluorescence, CD and NMR spectroscopies) and X-ray crystallography. We are particularly interested in the catalysis of reactions of nucleic acids (RNA and DNA) and most recently in the question of how structure, but not sequence, specific nucleases achieve specificity in nucleic acid hydrolysis.
Flap endonucleases (FENs, shown below human FEN1 (hFEN1) with DNA substrate) have been the focus of much of our recent work. FENs are a vital component of the lagging strand DNA replication apparatus in all organisms and also play role in DNA repair in eukaryotes. FENs remove 5’-single-stranded protrusions to double-stranded DNA known as flaps, formed as a result of DNA polymerase strand displacement synthesis. In humans FENs have to carry out approximately 50 million phosphate diester hydrolyses to allow replication of a single cell. FEN1 is the prototypical member of a superfamily of structure-specific 5’-nucleases whose differing activities span all the major DNA metabolic pathways.
Each of the 5’-nucleases acts on a different substrates. Recently, we have been trying to understand both common features of 5’-nucleases and the specialised mechanisms they use to carry out their extraordinary feats of molecular recognition. We have a learnt that the single stranded DNA flap is threaded through a hole in the protein known as the helical arch. This threading mechanism allows the enzyme to act on discontinuous flap DNAs that have free 5’-ends, but importantly not at single-strand double-strand junctions in DNA that are continuous. The DNA becomes threaded through the arch while this region of the protein is disordered. Forming the helical structure delivers key amino acid residues to the active site to catalyse phosphodiester hydrolysis.
Undergraduate and postgraduate taught modules
- Bennet I, Finger LD, Baxter NJ, Ambrose B, Hounslow A, Thompson MJ, Exell JC, Shahari NNBM, Craggs TD, Waltho JP & Grasby JA (2018) Regional conformational flexibility couples substrate specificity and scissile phosphate diester selectivity in human flap endonuclease 1. Nucleic Acids Research, 46(11), 5618-5633. View this article in WRRO
- Grasby JA, Thompson MJ, Gotham V & Ciani B (2018) A conserved loop-wedge motif moderates reaction site search and recognition by FEN1. Nucleic Acids Research. View this article in WRRO
- Tsutakawa SE, Thompson MJ, Arvai AS, Neil AJ, Shaw SJ, Algasaier SI, Kim JC, Finger LD, Jardine E, Gotham VJB, Sarker AH, Her MZ, Rashid F, Hamdan SM, Mirkin SM, Grasby JA & Tainer JA (2017) Corrigendum: Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability. Nature Communications. View this article in WRRO
- Shaw SJ, Finger LD & Grasby JA (2017) Human Exonuclease 1 Threads 5'-Flap Substrates Through its Helical Arch.. Biochemistry, 56(29), 3704-3707. View this article in WRRO
- Tsutakawa SE, Thompson MJ, Arvai AS, Neil AJ, Shaw SJ, Algasaier SI, Kim JC, Finger LD, Jardine E, Gotham VJB, Sarker AH, Her MZ, Rashid F, Hamdan SM, Mirkin SM, Grasby JA & Tainer JA (2017) Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability. Nature Communications, 8. View this article in WRRO
- Exell JC, Thompson MJ, Finger LD, Shaw SJ, Debreczeni J, Ward TA, McWhirter C, Siöberg CLB, Martinez Molina D, Abbott WM, Jones CD, Nissink WM, Durant ST & Grasby JA (2016) Cellularly active N-hydroxyurea FEN1 inhibitors block substrate entry to the active site. Nature Chemical Biology, 12, 815-821. View this article in WRRO
- Algasaier SI, Exell JC, Bennet IA, Thompson MJ, Gotham VJB, Shaw SJ, Craggs TD, Finger LD & Grasby JA (2016) DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction. Journal of Biological Chemistry, 291(15), 8258-8268. View this article in WRRO
- Kime L, Clarke JE, Romero A D, Grasby JA & McDowall KJ (2014) Adjacent single-stranded regions mediate processing of tRNA precursors by RNase E direct entry.. Nucleic Acids Res, 42(7), 4577-4589. View this article in WRRO
- Patel N, Exell JC, Jardine E, Ombler B, Finger LD, Ciani B & Grasby JA (2013) Proline Scanning Mutagenesis Reveals a Role for the Flap Endonuclease-1 Helical Cap in Substrate Unpairing. J. Biol. Chem., 288, 34239-34248. View this article in WRRO
- Finger LD, Patel N, Beddows A, Ma L, Exell JC, Jardine E, Jones AC & Grasby JA (2013) Observation of unpaired substrate DNA in the flap endonuclease-1 active site.. Nucleic Acids Res, 41(21), 9839-9847. View this article in WRRO
- Wilkinson OJ, Latypov V, Tubbs JL, Millington CL, Morita R, Blackburn H, Marriott A, McGown G, Thorncroft M, Watson AJ, Connolly BA, Grasby JA, Masui R, Hunter CA, Tainer JA, Margison GP & Williams DM (2012) Alkyltransferase-like protein (Atl1) distinguishes alkylated guanines for DNA repair using cation-π interactions.. Proc Natl Acad Sci U S A, 109(46), 18755-18760.
- Abdu K, Aiertza MK, Wilkinson OJ, Grasby JA, Senthong P, Povey AC, Margison GP & Williams DM (2012) Synthesis of oligodeoxyribonucleotides containing a conformationally-locked anti analogue of O6-methyl-2'-deoxyguanosine and their recognition by MGMT and Atl1.. Chem Commun (Camb), 48(91), 11214-11216.
- Beddows A, Patel N, Finger LD, Atack JM, Williams DM & Grasby JA (2012) Interstrand disulfide crosslinking of DNA bases supports a double nucleotide unpairing mechanism for flap endonucleases.. Chem Commun (Camb), 48(71), 8895-8897.
- Patel N, Atack JM, Finger LD, Exell JC, Thompson P, Tsutakawa S, Tainer JA, Williams DM & Grasby JA (2012) Flap endonucleases pass 5'-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5'-ends.. Nucleic Acids Res, 40(10), 4507-4519. View this article in WRRO
- Grasby JA, Finger LD, Tsutakawa SE, Atack JM & Tainer JA (2012) Unpairing and gating: Sequence-independent substrate recognition by FEN superfamily nucleases. Trends in Biochemical Sciences, 37(2), 74-84.
- Grasby JA, Finger LD, Tsutakawa SE, Atack JM & Tainer JA (2012) Unpairing and gating: sequence-independent substrate recognition by FEN superfamily nucleases.. Trends Biochem Sci, 37(2), 74-84.
- Tomlinson CG, Syson K, Sengerová B, Atack JM, Sayers JR, Swanson L, Tainer JA, Williams NH & Grasby JA (2011) 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, 286(35), 30878-30887.
- Tsutakawa SE, Classen S, Chapados BR, Arvai AS, Finger LD, Guenther G, Tomlinson CG, Thompson P, Sarker AH, Shen B, Cooper PK, Grasby JA & Tainer JA (2011) Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily.. Cell, 145(2), 198-211.
- Sengerová B, Tomlinson C, Atack JM, Williams R, Sayers JR, Williams NH & Grasby JA (2010) Brønsted analysis and rate-limiting steps for the T5 flap endonuclease catalyzed hydrolysis of exonucleolytic substrates.. Biochemistry, 49(37), 8085-8093.
- Tomlinson CG, Atack JM, Chapados B, Tainer JA & Grasby JA (2010) Substrate recognition and catalysis by flap endonucleases and related enzymes.. Biochem Soc Trans, 38(2), 433-437.
- Finger LD, Blanchard MS, Theimer CA, Sengerová B, Singh P, Chavez V, Liu F, Grasby JA & Shen B (2009) The 3'-flap pocket of human flap endonuclease 1 is critical for substrate binding and catalysis.. J Biol Chem, 284(33), 22184-22194.
- Syson K, Tomlinson C, Chapados BR, Sayers JR, Tainer JA, Williams NH & Grasby JA (2008) Three metal ions participate in the reaction catalyzed by T5 flap endonuclease.. J Biol Chem, 283(42), 28741-28746.
- Williams R, Sengerová B, Osborne S, Syson K, Ault S, Kilgour A, Chapados BR, Tainer JA, Sayers JR & Grasby JA (2007) Comparison of the catalytic parameters and reaction specificities of a phage and an archaeal flap endonuclease.. J Mol Biol, 371(1), 34-48.
- Bastock JA, Webb M & Grasby JA (2007) The pH-dependence of the Escherichia coli RNase HII-catalysed reaction suggests that an active site carboxylate group participates directly in catalysis.. J Mol Biol, 368(2), 421-433.
- Marsh AJ, Williams DM & Grasby JA (2004) The synthesis and properties of oligoribonucleotide-spermine conjugates.. Org Biomol Chem, 2(14), 2103-2112.
- Kovacheva YS, Tzokov SB, Murray IA & Grasby JA (2004) The role of phosphate groups in the VS ribozyme-substrate interaction.. Nucleic Acids Res, 32(21), 6240-6250.
- Sidorov AV, Grasby JA & Williams DM (2004) Sequence-specific cleavage of RNA in the absence of divalent metal ions by a DNAzyme incorporating imidazolyl and amino functionalities.. Nucleic Acids Res, 32(4), 1591-1601.
- Redko Y, Tock MR, Adams CJ, Kaberdin VR, Grasby JA & McDowall KJ (2003) Determination of the catalytic parameters of the N-terminal half of Escherichia coli ribonuclease E and the identification of critical functional groups in RNA substrates.. J Biol Chem, 278(45), 44001-44008.
- Tock MR, Frary E, Sayers JR & Grasby JA (2003) Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonuclease.. EMBO J, 22(5), 995-1004.
- Tzokov SB, Murray IA & Grasby JA (2002) The role of magnesium ions and 2'-hydroxyl groups in the VS ribozyme-substrate interaction.. J Mol Biol, 324(2), 215-226.
- Dickman MJ, Ingleston SM, Sedelnikova SE, Rafferty JB, Lloyd RG, Grasby JA & Hornby DP (2002) The RuvABC resolvasome.. Eur J Biochem, 269(22), 5492-5501.
- Patel D, Tock MR, Frary E, Feng M, Pickering TJ, Grasby JA & Sayers JR (2002) A conserved tyrosine residue aids ternary complex formation, but not catalysis, in phage T5 flap endonuclease.. J Mol Biol, 320(5), 1025-1035.
- Dervan JJ, Feng M, Patel D, Grasby JA, Artymiuk PJ, Ceska TA & Sayers JR (2002) 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, 99(13), 8542-8547.
- Ingleston SM, Dickman MJ, Grasby JA, Hornby DP, Sharples GJ & Lloyd RG (2002) Holliday junction binding and processing by the RuvA protein of Mycoplasma pneumoniae.. Eur J Biochem, 269(5), 1525-1533.
- Dickman MJ, Conroy MJ, Grasby JA & Hornby DP (2002) RNA footprinting analysis using ion pair reverse phase liquid chromatography.. RNA, 8(2), 247-251.
- Gourlain T, Sidorov A, Mignet N, Thorpe SJ, Lee SE, Grasby JA & Williams DM (2001) Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR.. Nucleic Acids Res, 29(9), 1898-1905.
- Lee SE, Sidorov A, Gourlain T, Mignet N, Thorpe SJ, Brazier JA, Dickman MJ, Hornby DP, Grasby JA & Williams DM (2001) Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity.. Nucleic Acids Res, 29(7), 1565-1573.
- Lee SE, Vyle JS, Williams DM & Grasby JA (2000) Novel syntheses of (Z)-alkene and alkane base-modified nucleosides. TETRAHEDRON LETT, 41(2), 267-270.
- Pickering TJ, Garforth S, Sayers JR & Grasby JA (1999) 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, 274(25), 17711-17717.
- Young KJ, Vyle JS, Pickering TJ, Cohen MA, Holmes SC, Merkel O & Grasby JA (1999) The role of essential pyrimidines in the hairpin ribozyme-catalysed reaction.. J Mol Biol, 288(5), 853-866.
- Pickering TJ, Garforth SJ, Thorpe SJ, Sayers JR & Grasby JA (1999) A single cleavage assay for T5 5'-->3' exonuclease: determination of the catalytic parameters forwild-type and mutant proteins.. Nucleic Acids Res, 27(3), 730-735.
- Holmes SC, Pearson ND & Grasby JA (1999) Synthesis and incorporation of a novel nucleoside phosphoramidite linker into oligonucleotides. CHEMISTRY OF NUCLEIC ACID COMPONENTS, 2, 119-123.
- Lee SE, Mignet N, Vyle JS, Whetton L, Grasby JA & Williams DM (1999) The synthesis of modified 5-(aminoalkyl)- and 5-(aminoalkenyl)uridine 5 '-triphosphates. CHEMISTRY OF NUCLEIC ACID COMPONENTS, 2, 35-38.
- Vyle JS, Williams NH & Grasby JA (1998) A novel solid support for synthesis of 2 ',3 '-cyclic phosphate terminated oligonucleotides. TETRAHEDRON LETT, 39(43), 7975-7978.
- Vyle JS, Young KJ & Grasby JA (1998) Incorporation of methylated pyrimidine analogues into RNA. TETRAHEDRON LETT, 39(28), 5093-5096.
- Young KJ, Gill F & Grasby JA (1997) Metal ions play a passive role in the hairpin ribozyme catalysed reaction.. Nucleic Acids Res, 25(19), 3760-3766.
- Thorogood H, Grasby JA & Connolly BA (1996) Influence of the phosphate backbone on the recognition and hydrolysis of DNA by the EcoRV restriction endonuclease. A study using oligodeoxynucleotide phosphorothioates.. J Biol Chem, 271(15), 8855-8862.
- Vyle JS, Young KJ, Jones S & Grasby JA (1996) Synthesis of uridine analogues to probe the functional group requirements of the hairpin ribozyme. COLLECTION OF CZECHOSLOVAK CHEMICAL COMMUNICATIONS, 61, S280-S282.
- Young KJ, Gill F & Grasby JA (1996) Mechanistic studies of the hairpin ribozyme. COLLECTION OF CZECHOSLOVAK CHEMICAL COMMUNICATIONS, 61, S268-S270.
- Grasby JA, Mersmann K, Singh M & Gait MJ (1995) Purine functional groups in essential residues of the hairpin ribozyme required for catalytic cleavage of RNA.. Biochemistry, 34(12), 4068-4076.
- GAIT MJ, GRASBY JA, KARN J, MERSMANN K & PRITCHARD GE (1995) SYNTHETIC RIBONUCLEOTIDE ANALOGS FOR RNA STRUCTURE-FUNCTION STUDIES. NUCLEOSIDES & NUCLEOTIDES, 14(3-5), 1133-1144.
- GRASBY JA, SINGH M, KARN J & GAIT MJ (1995) SYNTHESIS AND APPLICATIONS OF OLIGORIBONUCLEOTIDES CONTAINING N-4-METHYLCYTIDINE. NUCLEOSIDES & NUCLEOTIDES, 14(3-5), 1129-1132.
- GRASBY JA, PRITCHARD CE & GAIT MJ (1994) APPLICATIONS OF SYNTHETIC OLIGORIBONUCLEOTIDE ANALOGS IN STUDIES OF RNA STRUCTURE AND FUNCTION. P INDIAN AS-CHEM SCI, 106(5), 1003-1022.
- Pritchard CE, Grasby JA, Hamy F, Zacharek AM, Singh M, Karn J & Gait MJ (1994) Methylphosphonate mapping of phosphate contacts critical for RNA recognition by the human immunodeficiency virus tat and rev proteins.. Nucleic Acids Res, 22(13), 2592-2600.
- Grasby JA & Gait MJ (1994) Synthetic oligoribonucleotides carrying site-specific modifications for RNA structure-function analysis.. Biochimie, 76(12), 1223-1234.
- Grasby JA, Jonathan P, Butler G & Gait MJ (1993) The synthesis of oligoribonucleotides containing O6-methylguanosine: the role of conserved guanosine residues in hammerhead ribozyme cleavage.. Nucleic Acids Res, 21(19), 4444-4450.
- SEELA F, MERSMANN K, GRASBY JA & GAIT MJ (1993) 7-DEAZAADENOSINE - OLIGORIBONUCLEOTIDE BUILDING-BLOCK SYNTHESIS AND AUTOCATALYTIC HYDROLYSIS OF BASE-MODIFIED HAMMERHEAD RIBOZYMES. HELV CHIM ACTA, 76(5), 1809-1820.
- Grasby JA & Connolly BA (1992) Stereochemical outcome of the hydrolysis reaction catalyzed by the EcoRV restriction endonuclease.. Biochemistry, 31(34), 7855-7861.
Conference proceedings papers
- Finger LD, Bennet IA, Hounslow A, Exell JC, Baxter NJ, Waltho JP & Grasby JA (2015) The Catalytic Cycle of hFEN1 Requires Protein and DNA Conformational Changes, but Are They Rate-Limiting?. PROTEIN SCIENCE, Vol. 24 (pp 147-147)
- Osborne SD & Grasby JA (2005) Investigations into substrate analogues for Fen-1. Chemistry of Nucleic Acid Components, Vol. 7 (pp 347-350)
- Kovacheva YS, Tzokov SB, Murray IA & Grasby JA (2002) Kinetic studies of the Neurospora vs ribozyme. CHEMISTRY OF NUCLEIC ACID COMPONENTS, Vol. 5 (pp 352-355)
- Marsh AJ, Price C & Grasby JA (2002) Synthesis of polyamine-uridine conjugates by derivatisation at the 5-position. CHEMISTRY OF NUCLEIC ACID COMPONENTS, Vol. 5 (pp 241-244)
- Tzokov SB, Macdonald IK, Murray IA & Grasby JA (2002) Investigating catalytic RNA molecules. CHEMISTRY OF NUCLEIC ACID COMPONENTS, Vol. 5 (pp 231-240)
- McDonald IK, Holmes SC, Young KJ, Vyle JS, Pickering TJ & Grasby JA (1999) Functional group mutagenesis of the hairpin ribozyme. CHEMISTRY OF NUCLEIC ACID COMPONENTS, Vol. 2 (pp 306-309)
- Pickering TJ, Garforth SJ, Sayers JR & Grasby JA (1999) Mechanistic studies of a 5 ' nuclease from T5 bacteriophage. CHEMISTRY OF NUCLEIC ACID COMPONENTS, Vol. 2 (pp 161-167)