Award details

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

Reference	BB/K005359/1
Principal Investigator / Supervisor	Professor Bernard Connolly
Co-Investigators / Co-Supervisors
Institution	Newcastle University
Department	Inst for Cell and Molecular Biosciences
Funding type	Research
Value (£)	363,666
Status	Completed
Type	Research Grant
Start date	04/03/2013
End date	03/03/2016
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 DNA polymerases, the document submitted by James Chong deals mainly with archaea. DNA polymerases are essential components of all PCR and DNA sequencing systems, two technologies whose impact it is impossible to overstate. PCR underpins the biosciences, having applications in medicine, genetics, forensics and industrial biotechnology. DNA sequencing has become cheap enough to make entire genome sequences routine. It is predicted that sequencing demand will increase rapidly, notably in the area of human personalised medicine. Our proposal seeks to fully understand Pol-D, rather than to test and develop it for PCR/sequencing applications. However, with the increasing use and sensitivity of PCR and sequencing methods, the more polymerases that can be brought to bear, the more likelihood of success in future applications. The potential of Pol-D in PCR and sequencing has yet to be tested. However, Professor Connolly has a long standing link with Bioline, a London-based company, which supplies enzymes for PCR. Bioline have expressed an interest in trialling Pol-D in PCR-based applications, should the enzyme show initial favourable properties, further developments will follow. In this respect Professor Connolly's experience with archaeal Pol-B is instructive. A number of BBBSRC grants were awarded, mainly to investigate fundamental properties. However, during this work uracil-insensitive Pol-B variants were discovered, with general superior PCR performance and more specific applicability with amplification of old/damaged DNA (which contains excess uracil). This Pol-B mutant (V93Q) has been commercialised and is sold by Stratagene. It has been used be very many investigators and also been developed as a sensitive PCR-based reagent for determining uracil content in DNA. A second mutant (D215A, D279G) has low fidelity and has been used by a number of persons for error prone PCR. In a similar fashion, fundamental studies of Pol-D, may give derivatives with useful, and as yet unanticipated, properties.

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/K006630/1 Biochemical and genetic characterisation of DNA polymerase D, a novel archaeal replicative polymerase

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