The Metallobiochemistry of a Flap Endonuclease

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 and flap endonuclease is one of these vital enzymes. Without flap endonucleases life forms cannot exist. Understanding how flap endonuclease functions at the molecular level is the principle aim of this work.

Flap endonucleases (FENs) are metallonucleases that play a vital role in DNA replication and repair. Functional data demonstrate that the overall FEN reaction requires three magnesium ions, which interplay in an unprecedented manner to produce specificity and catalysis. In this application we seek to understand how this complex interplay contributes to chemical catalysis and substrate binding, testing a model we have proposed for these processes. In this model one metal contributes to chemical catalysis, another to substrate binding, whilst a third is involved in both processes. An important mechanistic question is whether a third catalytic metal ion, not present in substrate-free structures, resides within the FEN active. This will be addressed using a programme of mutagenesis of the active site carboxylates to ascertain whether this perturbs binding of the lower affinity metal ion. The possible role of a conserved active site tyrosine in moderation of the cooperative association of metal ions required for substrate binding and adjustment of the chemical reactivity of the FEN-DNA complex will be investigated. The metal ion response of the FEN reaction will be studied with alternative cofactors. In cases where metal ions produce the predicted reactivity trends, the overall reaction characteristics should be similar to those of magnesium ions and provide an important validation of this data. In other cases, where reactivity is lower than would be predicted, the metal ion response may differ explaining this lowered reactivity. Finally efforts to obtain a crystal structure of the FEN-metal ion-DNA complex will be supported by supplying a substrate analogue and characterising candidate mutant proteins for co-crystallography. Much of the debate over the mechanism of metallonucleases is concerned with the functional relevance of co-crystal structures. Thus a comparative evaluation of functional and structural data will be informative.