Award details

Unpair to Repair or Degrade-Structure Sensing Nucleases

Reference	BB/M00404X/1
Principal Investigator / Supervisor	Professor Jane Grasby
Co-Investigators / Co-Supervisors
Institution	University of Sheffield
Department	Chemistry
Funding type	Research
Value (£)	655,979
Status	Completed
Type	Research Grant
Start date	30/12/2014
End date	31/08/2018
Duration	44 months

Abstract

Flap endonuclease-1 (FEN1), essential for lagging-strand DNA replication in all organisms, is the prototypical member of the 5'-nuclease superfamily. FEN1 processes Okazaki fragments removing 5'-flaps by catalysing phosphodiester hydrolysis one nucleotide (nt) into a duplex. We explain this reaction specificity by a novel double nucleotide unpairing (DNU) mechanism. DNU of the duplex end allows the scissile bond to contact catalytic metal ions and in hFEN1 requires a structural motif known as the helical cap. We propose this mechanism is universal for the superfamily. Here, we aim to understand the role of DNU in FEN-family catalysis, with implications for substrate selection, regulation of these enzymes in vivo and therapeutic inhibition strategies. We will investigate whether control of DNU is related to biological substrate recognition by hFEN1. We will explore protein-DNA interactions required to activate hFEN1-DNU apparatus with implications for biological specificity. Extending our studies to other superfamily members, we will test whether DNU is a universal mechanism. We will ask whether DNU mediated by hEXO1 requires the helical cap and specific protein motifs that stabilize this. We will determine whether prevention of DNU through interactions with other hEXO1 specific domains in its extended C-terminus and/or protein modification is used to regulate EXO1 activity, thereby forming the basis of auto-inhibition strategies that are also used by other 5'-nuclease superfamily members. The processive 5'-exoribonucleases (XRNs) also conserve the FEN1 protein architecture and active site. We will probe whether XRNs use DNU to deal with RNA secondary structure. In addition, we will begin to unravel how processivity is achieved by the RNA hydrolyases testing the possibility that this is related to helical cap structural integrity. Finally, we will investigate strategies that could be universally applied to inhibit the superfamily by preventing DNU.

Summary

Genetic information is stored within deoxyribonucleic acid (DNA) in almost all organisms. DNA is composed of two polymer strands that are entwined to form a twisted ladder, known as the double helix. The rungs of the ladder are made up of a DNA alphabet A, C, T and G. These alphabet pieces, known as nucleotides, pair with each other according to special rules. A will pair only with a T in the opposite strand, and G only with a C. Thus each strand of the DNA molecule serves as a template to specify the sequence of nucleotides during duplication, or replication, of the complementary strand. When DNA becomes damaged, or is temporarily formed into a non-double helical structure (e.g. during replication), genetic information can be lost with life threatening consequences. To reinstate the genome to its double-helical form it is essential that it is precisely cut at high speed, removing the aberrant portion of DNA. The protein catalysts (enzymes) that mediate fast cutting reactions on DNA are called nucleases. However, like a child playing with scissors or knives, potentially nucleases are risky molecules to have in cells. Uncontrolled and imprecise cutting of DNA could lead to destruction of genetic information. Instead nucleases must carry out reactions with high precision in the correct place and on the right DNA molecule. Understanding how structure-sensing high precision nucleases function is important. Failure of any one of them to act, or the risk of action in the wrong place, is life threatening. Nucleases restore DNA back to its double helical form by cutting the DNA on one strand, sensing where the correct double helical duplex structure still exists. One group of these enzymes, known as the 5'-nuclease superfamily, look similar but act in many different situations on different incorrect DNA structures. We discovered that one member of the 5'-nuclease superfamily called FEN1 senses duplexes and carries out vital high precision cutting by testing the ability of the ends of double helical DNA to unpair. Specifically, FEN1 makes two nucleotides at the end of one strand of the intact duplex unravel so it reaches the nuclease active site and can get cut. This end of duplex sensing mechanism is known as double nucleotide unpairing (DNU). Unlike ends of duplexes, normal double helical DNA cannot undergo DNU and so is protected from nuclease damage. Moreover, the position of the active site relative to the nuclease bound duplex, controls which DNA strand is cut and precisely where it is incised. We plan to study the way FEN1 and other superfamily members carry out DNU. We have some evidence that the ability to carry out DNU is very precisely controlled by the 5'-nuclease superfamily in a way related to each enzyme's biological function. For FEN1, which is an essential nuclease during replication in all life forms, we will test if DNU ability is related to correct target DNA recognition. We also plan to explore whether the DNU mechanism is universal by testing another family member EXO1. We will ask whether controlling the ability to effect DNU by interaction with other proteins can be used as an on-off switch to control EXO1 so that it only acts when needed. Using the DNU apparatus may also be important to cut another nucleic acid known as ribonucleic acid (RNA). RNA is a single stranded molecule, but can fold up to contain double helical regions by base-pairing with itself. Unlike DNA, RNA is targeted for destruction by nucleases when it has served its purpose. We will ask whether DNU is used to deal with duplex regions in RNA molecules by an enzyme called XRN1. We will also ask whether preventing DNU is a way we can therapeutically prevent nuclease action with small molecules. Elevated amounts of nucleases are present in rapidly dividing cancer cells and some are also required for viral infection. Thus understanding how to interfere with DNU could lead to new treatments for human diseases.

Impact Summary

The impact of this work is directed towards: (1) Maintaining and enhancing the skills base of the biological and bioteconology workforce, by training and developing two PDRAs ensuring that the skills base exists for innovation and science that benefits the economy and society. (2) Pharmaceutical and biotechnology companies developing novel targets for drug design and seeking to inhibit 5'- superfamily members. FEN1 is already a target for therapeutic intervention for a number of companies, including some located within the UK. Other members of the FEN1 protein family are also potential drug targets. Ultimately such efforts rely on molecular understanding and effective high throughput assays, and several aspects of this proposal are therefore highly pertinent to inhibitor development and strategies towards inhibitor design. Ultimately society will benefit if new therapeutic approaches become available. (3) Biotechnologists seeking to develop tools for mutation detection or gene expressions. e.g. FENs are an integral part of the Taqman technology. Enzymes with subtly different activities could be useful for developing new assays and procedures. (4) XRN1 is commercially available for use in molecular biology (New England Biolabs). Enzymes with subtly different activities could be useful for specific applications and understanding the substrate preferences of this enzyme in more detail will assist those seeking to use XRN1 in their experiments.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search