Award details

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

Reference	BB/K006630/1
Principal Investigator / Supervisor	Professor James Chong
Co-Investigators / Co-Supervisors
Institution	University of York
Department	Biology
Funding type	Research
Value (£)	54,281
Status	Completed
Type	Research Grant
Start date	11/02/2013
End date	10/02/2016
Duration	36 months

Abstract

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.

Summary

The replication of chromosomal DNA is fundamental to all life, ensuring the accurate transmission of genetic information from parent to progeny. All living organisms evolved from a single common ancestor and extant life forms are classified into three large domains, bacteria, eukarya and archaea. In all three domains the co-ordinated activity of a large number of proteins, assembled as a multi-protein complex called the replisome, is used to bring about tightly regulated, rapid and accurate copying of DNA. DNA polymerases, the enzymes responsible for actual copying of DNA, are a key replisome component. In bacteria, the domain in which replication was first studied and still the best understood, a C-family DNA polymerase (DNA polymerase III) is used to copy DNA. However, the two other domains, eukarya and archaea lack homologues of Pol III, rather their genomes encode primarily family-B polymerases. In eukaryotes a pair of family-B enzymes are used to copy the two DNA strands. All archaea contain at least one family-B polymerase, with biochemical properties compatible with DNA replication. By analogy with eukaryotes, it has commonly been assumed that in archaea, the universally observed B-polymerase is responsible for replication. However, in a collaboration with scientists (Prof. John Reeve and Dr. Tom Santangelo) at Ohio State University, Professor Connolly has shown that removal of the single family-B polymerase from the archaeon Thermococcus kodakarensis (Tkod) is without influence. This Tkod deletion strain grows at the same rate as the wild type, has the same sensitivity to DNA damaging reagents and does not make more errors during DNA replication, suggesting Pol-B is not critical for DNA replication. A novel DNA polymerase, Pol-D, has been observed in four of the five characterised archaeal phyla (eury-, thaum-, kor- and nanoarchaea), although the enzyme appears to be missing from the fifth phylum, the crenarchaea. Pol-D also has properties compatible with DNA replication and, unlike, Pol-B, cannot be deleted in Tkod. These observations raise the possibility that, in most archaeal species, the family-D polymerase is responsible for copying DNA. The family-D polymerases are poorly characterised and appear unique in terms of sub-unit structure (a heterodimer consisting of a large, polymerase, sub-unit and a small, proof reading exonuclease, sub-unit) and have little amino acid similarity with other (family-B and -C) replicative polymerases. It is, therefore, proposed to thoroughly investigate the properties and functions of Pol-D using a combination of biochemical and genetic methods. We will purify the enzyme using gentle approaches that should preserve the metallo-cofactors (notably an Fe-S cluster, susceptible to destruction by oxygen) suspected to be present in the polymerase. A full set of in vitro experiments will be used to determine how the enzyme copies DNA and responds to DNA damage and it is also hoped to determine a high resolution structure. Complementary in vivo experiments, manipulating the chromosomal Pol-D genes in the genetically tractable archaeon Methanococcus maripaludis, will elucidate the role the enzyme plays in the cell. Demonstrating that most archaea use Pol-D for replication, and so that the three domains of life have a different replicative polymerase, raises profound questions about the evolution of DNA replication and the advantages of different replicative strategies.

Impact Summary

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.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/K005359/1 Biochemical and genetic characterisation of DNA polymerase D, a novel archaeal replicative polymerase

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