Enzymology and applications of thermophilic enzymes that replicate and manipulate nucleic acids

DNA replication is a fundamental process for all forms of life, ensuring accurate copying of the genome for the transmission of genetic information to progeny. DNA is copied by a DNA polymerase, which, in vitro, shows activity in the absence of other proteins. However, within cells the polymerase is always associated with other proteins, making up the replisome, a multi-protein machine responsible for DNA replication. Key proteins include: 1) PCNA, the sliding clamp that encircles DNA and prevents polymerase dissociation, thereby increasing processivity. 2) RPC, the clamp loader, responsible for loading PCNA onto DNA 3) RPA, single stranded binding protein responsible for preventing DNA reannealing. 4) GINS, a central complex that appears to assemble and hold all the replisome components together. The archaea, a third domain of life, are excellent organisms for the in vitro study of replication. Many organisms are hyperthermophilic, easing protein purification, and archaea serve as simplified models for eukaryotic replication. It is proposed to investigate the structure and function of replisome components with emphasis on the interplay between them and protein-protein interactions formed. A variety of techniques will be used including kinetic and binding methods, site-directed mutagenesis, low resolutions structural approaches (X-ray/neutron scattering and AUC) and high resolution X-ray crystallography. In this manner it is hoped to obtain a full understanding of how the archaeal replisome functions and develop systems capable of, in vitro, co-ordinated replication of double-stranded DNA. Thermophilic DNA polymerases are key reagents in the polymerase chain reaction (PCR), the most widely used technique in the biosciences. Furthermore, many of the replisome proteins, while not essential for in vitro DNA polymerase activity, may improve performance during the PCR. Thus the influence of these proteins, in a number of PCR applications, particularly real time PCR, will be evaluated.