Professor Jane A. Grasby
Department of Chemistry
Professor of Biological Chemistry
j.a.grasby@sheffield.ac.uk
+44 114 222 9478
+44 114 222 9478
Dainton Building
Full contact details
Professor Jane A. Grasby
Department of Chemistry
Dainton Building
13 Brook Hill
Sheffield
S3 7HF
Department of Chemistry
Dainton Building
13 Brook Hill
Sheffield
S3 7HF
- Profile
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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.
- Research interests
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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.
- Publications
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Journal articles
- Efficient synthesis of DNA duplexes containing reduced acetaldehyde interstrand cross-links. Journal of the American Chemical Society, 145(2), 953-959.
- Flap endonuclease 1 mutations A159V and E160D cause genomic instability by slowing reaction on double-flap substrates. Biochemistry, 57(50), 6838-6847. View this article in WRRO
- A conserved loop-wedge motif moderates reaction site search and recognition by FEN1. Nucleic Acids Research, 46(15), 7858-7872. View this article in WRRO
- 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
- Corrigendum: Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability. Nature Communications.
- Human Exonuclease 1 Threads 5'-Flap Substrates Through its Helical Arch.. Biochemistry, 56(29), 3704-3707. View this article in WRRO
- Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability. Nature Communications, 8. View this article in WRRO
- Cellularly active N-hydroxyurea FEN1 inhibitors block substrate entry to the active site. Nature Chemical Biology, 12, 815-821. View this article in WRRO
- 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
- 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
- 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
- Observation of unpaired substrate DNA in the flap endonuclease-1 active site.. Nucleic Acids Res, 41(21), 9839-9847. View this article in WRRO
- The Wonders of Flap Endonucleases: Structure, Function, Mechanism and Regulation, 301-326. View this article in WRRO
- 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.
- 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.
- Interstrand disulfide crosslinking of DNA bases supports a double nucleotide unpairing mechanism for flap endonucleases.. Chem Commun (Camb), 48(71), 8895-8897.
- 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
- Unpairing and gating: Sequence-independent substrate recognition by FEN superfamily nucleases. Trends in Biochemical Sciences, 37(2), 74-84.
- Unpairing and gating: sequence-independent substrate recognition by FEN superfamily nucleases.. Trends Biochem Sci, 37(2), 74-84.
- 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.
- Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily.. Cell, 145(2), 198-211.
- Brønsted analysis and rate-limiting steps for the T5 flap endonuclease catalyzed hydrolysis of exonucleolytic substrates.. Biochemistry, 49(37), 8085-8093.
- Substrate recognition and catalysis by flap endonucleases and related enzymes.. Biochem Soc Trans, 38(2), 433-437.
- The 3'-flap pocket of human flap endonuclease 1 is critical for substrate binding and catalysis.. J Biol Chem, 284(33), 22184-22194.
- Three metal ions participate in the reaction catalyzed by T5 flap endonuclease.. J Biol Chem, 283(42), 28741-28746.
- Comparison of the catalytic parameters and reaction specificities of a phage and an archaeal flap endonuclease.. J Mol Biol, 371(1), 34-48.
- 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.
- The synthesis and properties of oligoribonucleotide-spermine conjugates.. Org Biomol Chem, 2(14), 2103-2112.
- The role of phosphate groups in the VS ribozyme-substrate interaction.. Nucleic Acids Res, 32(21), 6240-6250.
- 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.
- 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.
- Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonuclease.. EMBO J, 22(5), 995-1004.
- The role of magnesium ions and 2'-hydroxyl groups in the VS ribozyme-substrate interaction.. J Mol Biol, 324(2), 215-226.
- The RuvABC resolvasome.. Eur J Biochem, 269(22), 5492-5501.
- A conserved tyrosine residue aids ternary complex formation, but not catalysis, in phage T5 flap endonuclease.. J Mol Biol, 320(5), 1025-1035.
- 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.
- Holliday junction binding and processing by the RuvA protein of Mycoplasma pneumoniae.. Eur J Biochem, 269(5), 1525-1533.
- RNA footprinting analysis using ion pair reverse phase liquid chromatography.. RNA, 8(2), 247-251.
- 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.
- Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity.. Nucleic Acids Res, 29(7), 1565-1573.
- Mutagenesis of conserved residues in bacteriophage T5 5′-3′ exonuclease support model of separate mechanisms of endo- and exonucleolytic cleavage. Biochemical Society Transactions, 29(1), A30-A30.
- Novel syntheses of (Z)-alkene and alkane base-modified nucleosides. TETRAHEDRON LETT, 41(2), 267-270.
- 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.
- The role of essential pyrimidines in the hairpin ribozyme-catalysed reaction.. J Mol Biol, 288(5), 853-866.
- 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.
- Synthesis and incorporation of a novel nucleoside phosphoramidite linker into oligonucleotides. CHEMISTRY OF NUCLEIC ACID COMPONENTS, 2, 119-123.
- The synthesis of modified 5-(aminoalkyl)- and 5-(aminoalkenyl)uridine 5 '-triphosphates. CHEMISTRY OF NUCLEIC ACID COMPONENTS, 2, 35-38.
- A novel solid support for synthesis of 2 ',3 '-cyclic phosphate terminated oligonucleotides. TETRAHEDRON LETT, 39(43), 7975-7978.
- Incorporation of methylated pyrimidine analogues into RNA. TETRAHEDRON LETT, 39(28), 5093-5096.
- Metal ions play a passive role in the hairpin ribozyme catalysed reaction.. Nucleic Acids Res, 25(19), 3760-3766.
- 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.
- Mechanistic studies of the hairpin ribozyme. COLLECTION OF CZECHOSLOVAK CHEMICAL COMMUNICATIONS, 61, S268-S270.
- Synthesis of uridine analogues to probe the functional group requirements of the hairpin ribozyme. COLLECTION OF CZECHOSLOVAK CHEMICAL COMMUNICATIONS, 61, S280-S282.
- Purine functional groups in essential residues of the hairpin ribozyme required for catalytic cleavage of RNA.. Biochemistry, 34(12), 4068-4076.
- SYNTHESIS AND APPLICATIONS OF OLIGORIBONUCLEOTIDES CONTAINING N-4-METHYLCYTIDINE. NUCLEOSIDES & NUCLEOTIDES, 14(3-5), 1129-1132.
- SYNTHETIC RIBONUCLEOTIDE ANALOGS FOR RNA STRUCTURE-FUNCTION STUDIES. NUCLEOSIDES & NUCLEOTIDES, 14(3-5), 1133-1144.
- APPLICATIONS OF SYNTHETIC OLIGORIBONUCLEOTIDE ANALOGS IN STUDIES OF RNA STRUCTURE AND FUNCTION. P INDIAN AS-CHEM SCI, 106(5), 1003-1022.
- 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.
- Synthetic oligoribonucleotides carrying site-specific modifications for RNA structure-function analysis.. Biochimie, 76(12), 1223-1234.
- The synthesis of oligoribonucleotides containing O6-methylguanosine: the role of conserved guanosine residues in hammerhead ribozyme cleavage.. Nucleic Acids Res, 21(19), 4444-4450.
- Synthesis and applications of oligoribonucleotides containing guanosine analogues. Collection of Czechoslovak Chemical Communications, 58(s1), 154-157.
- 7-DEAZAADENOSINE - OLIGORIBONUCLEOTIDE BUILDING-BLOCK SYNTHESIS AND AUTOCATALYTIC HYDROLYSIS OF BASE-MODIFIED HAMMERHEAD RIBOZYMES. HELV CHIM ACTA, 76(5), 1809-1820.
- Stereochemical outcome of the hydrolysis reaction catalyzed by the EcoRV restriction endonuclease.. Biochemistry, 31(34), 7855-7861.
- ChemInform Abstract: 7-Deazaadenosine: Oligoribonucleotide Building Block Synthesis and Autocatalytic Hydrolysis of Base-Modified Hammerhead Ribozymes.. ChemInform, 24(48), no-no.
Chapters
- The hairpin ribozyme In Meunier B (Ed.) (pp. 295-306).
- Nucleotides and nucleic acids, Organophosphorus Chemistry (pp. 161-230). Royal Society of Chemistry
- Nucleotides and nucleic acids, Organophosphorus Chemistry (pp. 170-236). Royal Society of Chemistry
- Nucleotides and nucleic acids, Organophosphorus Chemistry (pp. 177-263). Royal Society of Chemistry
- Nucleotides and nucleic acids, Organophosphorus Chemistry (pp. 196-264). Royal Society of Chemistry
Conference proceedings papers
- The Catalytic Cycle of hFEN1 Requires Protein and DNA Conformational Changes, but Are They Rate-Limiting?. PROTEIN SCIENCE, Vol. 24 (pp 147-147)
- Defining the Superfamily Conserved Mechanism for Flap Endonucleases FEN1 and XPG Specificity for 5' Flap DNA and DNA Bubbles, Respectively, by Hybrids Methods of Crystallography, SAXS, EM, and Computation. Biophysical Journal, Vol. 106(2) (pp 230a-230a)
- Investigations into substrate analogues for Fen-1. Chemistry of Nucleic Acid Components, Vol. 7 (pp 347-350)
- Kinetic studies of the Neurospora vs ribozyme. CHEMISTRY OF NUCLEIC ACID COMPONENTS, Vol. 5 (pp 352-355)
- Investigating catalytic RNA molecules. CHEMISTRY OF NUCLEIC ACID COMPONENTS, Vol. 5 (pp 231-240)
- Synthesis of polyamine-uridine conjugates by derivatisation at the 5-position. CHEMISTRY OF NUCLEIC ACID COMPONENTS, Vol. 5 (pp 241-244)
- Mechanistic studies of a 5 ' nuclease from T5 bacteriophage. CHEMISTRY OF NUCLEIC ACID COMPONENTS, Vol. 2 (pp 161-167)
- Functional group mutagenesis of the hairpin ribozyme. CHEMISTRY OF NUCLEIC ACID COMPONENTS, Vol. 2 (pp 306-309)
Website content
- Home Page.
- Efficient synthesis of DNA duplexes containing reduced acetaldehyde interstrand cross-links. Journal of the American Chemical Society, 145(2), 953-959.
- Teaching interests
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Organic Chemistry; Biological Chemistry
- Teaching activities
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Undergraduate and postgraduate taught modules
- Carbonyl Compounds and Carboxylic Acids (Level 1)
This lecture course introduces methods for the synthesis of carboxylic acid derivatives and discusses their reactions. - Chemistry in a Sustainable Future (Level 1)
- Introduction to Chemical Biology & Medicinal Chemistry (Level 3)
This course introduces the basic principles of chemical biology and medicinal chemistry - Skills for Success (Level 3; course Leader)
The Skills for Success Project aims to ensure that students identify and develop skills that will be of use to them in life, future study and employment and provide a basis from which they can undertake further training of a professional nature.
Support Teaching:
- Tutorials: Level 1 General Chemistry.
- Level 3 Literature Review
Laboratory Teaching:
- Level 4 Research Project
- Carbonyl Compounds and Carboxylic Acids (Level 1)