Biochemical and genetic characterisation of DNA polymerase D, a novel archaeal replicative polymerase

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

A combination of genetic and biochemical approaches will be used to elucidate the function, role, features and properties of the family-D DNA polymerase, a unique enzyme confined to the majority of archaeal phyla. Current evidence, far from complete or conclusive, suggests that Pol-D: 1) is likely the major replicative polymerase in the archaea where it is present; 2) is a metallo-enzyme that contains Zn and an Fe-S cluster; 3) is strongly inhibited by uracil during replication. Pol-D appears unique in terms of composition (dimeric with a large polymerase and a small proof reading sub-unit) and its amino acid sequence has little in common with other replicative (family-A in some viruses, family-B in eukaryotes, family-C in bacteria) or repair polymerases. A full investigation of Pol-D requires preparation in a metallo-competent from, particularly in regard to labile Fe-S clusters. Thus the protein will be prepared by heterologous overexpression of protein in a genetically tractable archaeal host Methanococcus maripaludis with purification under anaerobic conditions. These precautions are essential for preserving Fe-S centres. The metal ion cofactors in Pol-D will be fully characterised and biochemical experiments will elucidate how the polymerase copies DNA and interacts with the damaged base uracil. Both high (X-ray crystallography) and low (analytical ultracentrifugation, small angle X-ray scattering) resolution methods will be used to obtain structural information. Key amino acids (including the cysteines that serve as metal ligands) will be probed by mutagenesis. These experiments will be complemented by genetic approaches using Methanococcus maripaludis. Mutations will be introduced into the chromosomal genes that encode the Pol-D sub-units to change critical amino acids and the phenotype recorded. The complementary biochemical and genetic approaches should lead to a full understanding of Pol-D.

The science proposed in this application is primarily "fundamental" in nature, aiming to elucidate the features and function of a novel archaeal DNA-polymerase, Pol-D. While it is not explicitly planned to carry out "applied" research i.e. to develop reagents/processes with a commercial application in mind, the work impinges in two areas of huge general relevance, DNA polymerases and genetic manipulation of the archaea. Both the PI (Connolly, Newcastle) and the co-investigator (Chong, York) will be involved in all impact activities that arise from this grant but Connolly will lead with DNA polymerases and Chong with archaeal genetic modification. Therefore, this statement concentrates on archaea while the document submitted by Bernard Connolly deals mainly with DNA polymerases.

While no direct commercial products are anticipated, there are enormous potential indirect outputs with very large commercial and societal significance. The ability to genetically manipulate the bacterial and eukaryotic domains of life has impinged on everyone, e.g. easier availability of therapeutic proteins such as insulin and blood clotting factors, through to genetically modified plants. However, exploitation of the archaeal domain using genetic technology has hardly begun. Yet this domain has interesting and useful properties such as exceedingly high thermostability, through to the production of potential bio-fuels such as hydrogen and methane. While this application does not directly seek to genetically modify the archaea to humankinds advantage, it should improve the tools that allow others to do so.

Genetic technologies for the archaea lag far behind those available for bacteria and eukaryotes, greatly limiting the uses of this domain. However, genetic methods for the archaea are slowly emerging and it is at last becoming possible to use these organisms in applications routine with genetically engineered bacteria and eukaryotes (e.g. protein overexpression, drug production, bioremediation, generation of biofuels and crop improvement). While the work proposed in the application does not directly seek to genetically modify any archaea for a defined immediate commercial application, it does propose to improve and develop genetic technologies for the mesophilic archaeon Methanococcus maripaludis. This is one of the few archaea currently amenable to genetic modification. Additionally, most other tractable archaea are either extremophiles (grow at high temperatures) or halophiles (have extreme cellular salt concentrations) and therefore, in contrast to M. maripaludis, are generally unsuitable for expressing useful eukaryotic and bacterial proteins. M. maripaludis is a strict anaerobe, making it ideal for the purpose outlined in this application (the expression of a protein containing a putative Fe-S cluster likely to be highly oxygen sensitive). Adding to the genetic toolbox available for use in M. maripaludis is, therefore, of long-term interest to persons interested in exploiting the genetic potential of the archaeal domain, and particularly in developing tools for the expression of redox-sensitive proteins with applications in biofuel and fertilizer generation such as hydrogenases, components of the cellulosome and nitrogenases. We believe that providing tools for genetically manipulating the archaea represents an important and novel technology development in the biosciences, one of the BBSRCs strategic priorities.