Conformation, Reactivity, Dynamics and Selectivity of FEN Family Proteins Relevant to Replication and Repair.

Lead Research Organisation: University of Sheffield
Department Name: Chemistry


The ability of a cell to accurately duplicate the vast quantity of genetic information carried in its deoxyribonucleic acid (DNA) is critical to the survival of all living 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 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. The process of replicating our genetic material is awe-inspiring in its complexity, as it involves copying billions of nucleotides with exceptional speed and accuracy. This amazing feat is performed by a group of proteins that together form a "replication machine". Understanding the function of each of these proteins is critical since failure of any one of them may result in a life threatening disease. In addition to the problem of accurate replication, DNA is under continual onslaught from environmental chemicals and radiation (mainly the sun's rays). These alter the DNA bases so that they no longer pair up, as they should, again a life-threatening situation. Biological systems have evolved a number of enzymes (biological catalysts) that are involved in repairing damaged DNA. An enzyme involved in both DNA replication and repair is Flap endonuclease. Without flap endonucleases and enzymes like them (known as a family of proteins) life cannot exist. Understanding how flap endonuclease and its other family members function at the molecular level is the principle aim of this work. Furthermore, the information garnered here would be invaluable for those who seek to develop new cancer therapeutics by creating inhibitors of flap endonucleases, as large amounts of flap endonucleases are present in cancer cells and its abundance in cancer cells allows cancers to progress more rapidly. Flap endonucleases are specifically involved in the removal of DNA 'whiskers' that form in two specialized processes; one in replication and one in repair. These whiskers of DNA have to be removed with extraordinary precision; otherwise, DNA replication and repair will be faulty and could result in cancer. Flap endonucleases are the molecular scissors that remove the whiskers with the necessary precision. Recently, we have obtained 'snap-shots' of the protein in various conformations or 3D structures (i.e., a picture of scissors in an open and a closed state), and this information has greatly increased our understanding of flap endonucleases and its family members. Unfortunately, snap-shots of various conformations do not tell the entire story. Just as scissors must move to be an effective tool for cutting, flap endonucleases must be able to move its parts to cut the DNA whiskers. We have proposed to use a specialized technique called nuclear magnetic resonance, which can probe the magnetic properties of atoms, to quantify the motions of the proteins. An understanding of the motions of flap endonucleases will solidify our understand of flap endonucleases and its family members and help guide pharmaceutical drug discover and design programs.

Technical Summary

Flap endonucleases (FENs), vital to replication and repair, remove RNA and DNA flaps. Other FEN-like proteins, EXO1, GEN1 and XPG, act on other aberrant DNAs during repair and recombination. Together they represent a major class of related structure-specific nucleic acid hydrolysing enzymes. The critical nature of FENs, coupled to their overexpression in cancer cells, makes them an oncology target. We propose that both protein and DNA conformational changes are essential to FEN and related enzyme function. Firstly, the hallmark of the 5'-nuclease superfamily is reaction one nucleotide into a DNA duplex, albeit contained within a more complex nucleic acid structure. This is explained by double nucleotide unpairing (DNU) of the duplex termini, which allows only the scissile bond to enter the active site. Secondly, an extensive protein disorder-order transition occurs upon binding substrate. This protein conformational change assembles the active site to capture DNU residues. More than 2.5 nm away, it also fashions a separate binding site for the specific feature of FEN1 substrates, a single nucleotide 3'-flap. Whether these protein disorder-order transitions occur independently or in a coordinated fashion is unknown, as is their relationship to DNU. Here we propose to investigate FEN protein and substrate dynamics by measuring the rates of their respective conformational changes and the extent to which they are coupled using NMR ModelFree formalism and relaxation dispersion techniques. Furthremore, we will also use NMR dynamics measurements to assess the roles of specific residues highlighted by our recent structural work. The properties of hFEN1 proteins and substrates will be compared to another superfamily member hEXO1. Overall, we aim to understand the role of biomolecular dynamics in FEN family catalysis, with implications for regulation of these enzymes in vivo and therapeutic inhibition strategies.

Planned Impact

The immediate beneficiary will be the researcher CoI who will develop their research skills and be exposed to a multidisciplinary project.

Pharmaceutical and biotechnology companies seeking to inhibit 5'-superfamily members will directly benefit form this research. FEN is already a target for therapeutic intervention for a number of companies, including some located within the UK. Ultimately such efforts rely on molecular level understanding of structure and mechanism and several aspects of this proposal are highly pertinent to inhibitor development.

Our study will also impact other academic groups seeking to understand DNA replication and repair as it will define the features that give rise to specificity in this superfamily.

Longer term should our efforts result in a treatment it will increase UK pharmaceutical and biotechnology companies competitiveness and benefit society.
Description Flap endonuclease 1 acts on flapped DNA, that is DNA with protruding 5'- and 3'- single strands that need to be removed to complete replication. We used multidimensional NMR to show that the regions of FEN1 protein involved in 3'- and 5'-flap recognition (the a4-a5 arch and a2-a3 loop) are intrinsically disordered without DNA present. In the presence of DNA, the timescale of their motions is slowed, implying protein conformational change in response to substrate. A combination of conformational selection and induced fit in the protein arch region are used to progressively narrow the conformational ensemble distribution until the catalytically-competent state is reached. These results explain several of the puzzles of FEN1 mechanism and catalysis. Notably, significant controversy surrounded how FEN1 accommodates 5'-flaps within the arch, as DNA does not normally travel through holes in proteins without a coupled energy source like ATP hydrolysis. However, a disordered archway allows the easy passage of ssDNA and even flaps with some secondary structure, providing the required flexibility associated with various substrates. When the arch is ordered, it forms two a-helices (helical arch); this helical arch conformation positions the active site basic residues for catalysis and other basic residues for DNA recognition, allowing the scissile phosphate ester to transfer to the active site and reaction to take place. Thus, disorder-order in response to the appropriate structural features of the substrate couples recognition and catalysis. Moreover, combining protein conformational selection (i.e, part of the arch already transiently adopts the helical conformation) and induced fit allows for fast complex assembly and high specificity without producing very high affinity for product nicked DNA, thereby allowing for rapid turnover. Most recently we have also examined the conformational change using single molecule biophysics which has provided information about the changes in protein conformation with and without DNA.
Exploitation Route Of relevant to development of FEN1 inhibitors
Sectors Pharmaceuticals and Medical Biotechnology