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.
We are currently producing constructs and transforming them into M. maripaludis for protein expression.
|Effective start/end date
|11/02/13 → 10/02/16
- BBSRC (BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL): £54,281.00