A molecular mechanism for flap endonucleases and the 5' nuclease superfamily

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'. Flap endonucleases are part of this apparatus. 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. In addition to normal replication flap endonucleases and some other proteins like flap endonucleases are a group of these vital enzymes involved in repair. Without flap endonucleases and enzymes like them life cannot exist. Understanding how flap endonuclease and its other family members function at the molecular level is the principle aim of this work. This could lead to new treatments to human diseases, as large amounts of flap endonucleases are present in cancer cells. We plan to study whether flap endonucleases and other enzymes remodel the end of the DNA, unpairing it to react. This is important to understand as it may help with design of inhibitors that are specific. Vital to the process of replication is that flap endonucleases cut DNA at a specific place. We seek to understand how this happens and how cuts in the wrong place are avoided. Flap endonucleases contain a hole through which DNA is proposed to thread. This hole looks like an arch, but there is evidence that sometimes this part of the protein does not exist as an arch. We wish to understand under what circumstances the arch is formed and whether arch formation is required for the enzyme to do its job. This has implications for the types of DNA substrates it can act upon. We are going to contrast the behavior of FEN with other family members that also repair DNA to work out if they operate in the same way.

Flap endonucleases (FENs), the prototypical member of the 5'-nuclease superfamily are a critical component of DNA replication and repair. Other FEN-like proteins, EXO1, GEN1 and XPG, act during different repair processes and recombination. Together they represent a major class of essential divalent metal ion dependent structure specific nucleic acid hydrolysing enzymes. In this application we seek to probe the molecular mechanism of FENs and other family members provoked by recent structural information. We propose a mechanism for the FEN superfamily reaction, where for reaction to proceed the terminal two base pairs of the DNA duplex must be unpaired to move into the active site to bind to essential cofactors. We will test this mechanism. We will test the roles of new features of FENs indentified by our structures, namely the acid block that we suggest enforces cellular specificity and the N-terminus that we suggest plays a role in unpairing. We will establish whether protein disorder order transitions of the helical arch are a feature of FEN catalysis and define the features that influence this. Finally we seek to further understand the substrate requirements of XPG,with a view to more detailed understanding of XPG and GEN1 substrate specificities. Overall we have the goal of a molecular mechanism for FEN and family members.

The impact of this work is directed towards: (1) Pharmaceutical and biotechnology companies developing novel targets for drug design and seeking to inhibit 5'-superfamily members. 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 understanding and several aspects of this proposal are highly pertinent to inhibitor development. (2) Biotechnologists seeking to develop tools for mutation detection in DNA. e.g. FENs are an integral part of the Taqman mutation detection technology and Third Waves Technologies Invader genotyping assays. T5FEN is commercially available for use in cleaning up plasmid preps (Epicentre biotechnologies). Enzymes with subtly different activities could be useful for developing new assays and procedures.