Chromosomal DNA replication requires the complex interplay of a large number of essential and non-essential protein factors in a temporally- and spatially-coordinated manner. Determining how these factors act together to replicate the genome is central to understanding how the integrity of the genome is maintained within, and across, generations and how genetic diseases such as cancer in humans are avoided. The components of the replication machinery are also potential targets for anti-proliferative drugs and can be used as diagnostic markers for the proliferative state.
The complexity of the replication machinery favours the use of simple model systems to dissect problems of protein structure, function and regulation. Indeed, much of what we know about the eukaryotic replication apparatus has come from model system studies. In the MacNeill lab, research is primarily focused on dissecting the molecular biology of chromosomal DNA replication and genome stability using two contrasting genetically-tractable model systems, the eukaryotic fission yeast Schizosaccharomyces pombe and the halophilic archaeon Haloferax volcanii. In addition to this, we also study the molecular biology of T5-like bacteriophages (Demerecviridae), DNA ligase enzymes encoded by diverse bacteriophages and viruses, and carbohydrate-processing enzymes in haloarchaea. We use a variety of methods to address questions of protein structure and function within the chromosome replication apparatus, including genetics and molecular biology, biochemistry, structural biology and bioinformatics.
Chromosomal DNA replication in eukaryotes
DNA polymerase delta plays essential roles in eukaryotic chromosomal DNA replication and also in various DNA repair pathways. We are interested in understanding how Pol delta activity in regulated in vivo. Using the fission yeast Schizosacharomyces pombe as a model system, we are investigating how post-translational modifications affect Pol delta function in vivo, with particular emphasis on modification of the PolD3 and PolD4 subunits of the complex. We have also expressed and purified the four-subunit Pol delta complex from the thermophilic ascomycete fungus Chaetomium thermophilum (Ct) and have determined its structure by cryo-EM in collaboration with Dr Ramasubramanian Sundaramoorthy (University of Dundee, work in progress). We have also used X-ray crystallography to obtain an understanding of how Chaetomium thermophilum PCNA interacts with Ct PolD3, Ct PolD4 and the flap endonuclease Ct Fen1 at atomic resolution (collaboration with Dr Magnus Alphey, University of St Andrews).
Chromosomal DNA replication and repair in archaea
Archaea constitute the third domain of life on Earth, comprise an estimated 20% of the planet’s biomass and make major impacts on both biosphere and atmosphere. We are interested in understanding the enzymes and mechanisms of chromosomal DNA replication and repair in archaeal cells and in what this can tell us about the fundamental processes underlying the maintenance of genetic integrity in all cells. We use the highly tractable halophilic (salt-loving) archaeal organisms Haloferax volcanii and Haloarcula hispanica as model systems as this provides us with a range of molecular genetic tools for functional analysis in vivo. Previously we have studied single-stranded DNA binding proteins, the sliding clamp PCNA, ATP- and NAD-dependent DNA ligases, MCM helicase and an entirely novel DNA repair factor named NreA. We are currently investigating the cellular functions of the four Haloferax volcanii RecJ-like proteins to gain fundamental insights into how these proteins have evolved to safeguard the integrity of the archaeal genome.
Molecular biology of bacteriophage T5 replication
The T5-like bacteriophages (Demerecviridae) are a family of lytic bacteriophages that infect Gram-negative bacteria. The phage particles consist of an icosahedral capsid, a lengthy non-contractile tail and a double-stranded DNA genome with the capacity to encode ~160-180 proteins. A number of these proteins have been identified as being essential for phage DNA replication including a DNA polymerase, primase, DNA ligase and exonuclease. We are interested in understanding the function of the remaining essential proteins and understanding how these interact with one another to facilitate rapid replication of the phage genome during infection. We are characterising individual phage T5 proteins biochemically while at the same time using CRISPR/Cas genome editing methods to allow us to investigate in vivo function in greater detail. One key target is the DNA ligase encoded by T5-like phages. We have solved the structure of this enzyme (from phage vB_PreS_PR1 DNA, a collaboration with structural biologist Dr Julia Richardson, University of Edinburgh), revealing novel features of DNA ligase biology.
DNA ligases for biotech applications
DNA ligases are essential enzymes in all forms of life and are a cornerstone of recombinant DNA technology. The market leader New England Biolabs alone markets six different ligases in 15 different formulations. In this project, we are exploring the outer limits of DNA ligase sequence space to identify novel enzymes with enhanced properties with commercial potential. Building on recent phylogenetic analysis, we are focusing on highly diverged and previously unstudied ATP-dependent ligase enzymes encoded by crAss-like phages present in the human gut, by viruses that infect unicellular green algae and amoeba, and by early-branching kinetoplastids. Ligase proteins are being expressed and purified in recombinant form and the purified enzymes tested for stability and activity on different substrates under a range of conditions.