Choreography of eukaryotic chromosome replication

The human body contains over a thousand billion different cells, each created by cell division, that productively interact to make the tissues and organs of the human body. All complex animals and plants are made of numbers of different cell types whereas simpler forms of life, such as bacteria and yeast, comprise single cells. All cell types including bacteria, yeast and human cells use the same basic mechanisms to replicate themselves to generate more cells. Perhaps the most important component of each cell is its DNA, which contains the blueprint to make the cell (for example a brain cell, blood cell, or yeast cell). For this reason, the mechanisms used to replicate cellular DNA are among the most important aspects of cell biology. It is important that DNA is replicated properly each time because mistakes (mutations) can change the properties of the cell making the cell misbehave, for example in cancer, or cause the cell (and organism) to die.

Many machines interact to replicate DNA and the interactions need to be carefully controlled and coordinated in order that DNA is replicated properly. The machines that replicate DNA are perhaps analogous to those that individual multi-component parts of a car, such as engine, gears, clutch, brake, accelerator and steering wheel. In a car all these complex sub-components interact and coordinate to make the car drive as required. If individual parts of a car fail, or coordination fails (for example between clutch and accelerator) the car will most likely not work.

The human genome is 3 billion base pairs and each cell in the body contains this number of bases. A single mistake in copying any of the three billion bases has the potential to be harmful, perhaps most recognisably if the single mistake contributes to causing cancer. However, given the magnitude of the task of replicating the entire cellular DNA content, it is inevitable that mistakes are made. Therefore to help replicate DNA with highest fidelity possible cells have evolved numerous mechanisms to check for errors. When errors are detected a number of mechanisms can slow, stop or reverse replication while errors are corrected.

We have used simple yeast cells, a powerful model genetic system, to investigate how DNA replication is coordinated. These yeast cells are also the type that mankind has cultivated for thousands of years to make bread, wine and beer. We have engineered these cells and reduced the ability of the cells to replicate their DNA and then screened to identify the pathways and processes that respond to these defects. It is likely that similar defects are important during human ageing processes or can be induced in nature by drugs such as antibiotics or antifungals.

Our experiments on yeast cells with defective DNA replication have allowed us to identify mechanisms that help cells cope with failures in DNA replication. We identified a number of interesting interactions that give insight into how the machinery of replication is regulated. We will now use powerful molecular and cellular biology methods to understand the molecular and biochemical basis of interactions we have identified.

Eukaryotic DNA replication requires the coordination of scores of different protein, DNA and RNA species. To understand how replication is coordinated we have performed genome-wide genetic screens to identify second site mutations that enhance or suppress defects in each of the three essential DNA polymerases. Our screens highlight important roles for Replication Factor C (RFC) proteins, telomere binding proteins and nonsense mediated mRNA decay pathways in distinct aspects of DNA replication.

RFC is a five-protein complex that forms the clamp loader that loads the sliding clamp (PCNA) onto DNA. PCNA is a trimeric ring protein that slides along DNA and tethers polymerases to DNA. In eukaryotes the large subunit of RFC, Rfc1, can be replaced. Our data suggests that competition between different RFC complexes (containing Rfc1, Ctf18, Elg1 or Rad24) is important for efficient replication and more specifically that alternative RFCs are differently important for Pol alpha dependent replication initiation versus Pol delta and Pol epsilon dependent elongation. We will investigate the roles of the different RFC proteins using a variety of molecular and genetic approaches.

Rif1 and Rif2 play important roles in telomere length regulation. Our data shows that Rif1 but not Rif2 is critically important to yeast strains defective in Pol alpha or with telomere defects (cdc13-1) but is much less important for Pol delta and Pol epsilon defective strains. We aim to better understand the role of Rif1 in replication.

We have shown that nonsense mediated mRNA decay pathways (NMD) are critically important in strains defective in Pol alpha. We will test the hypothesis that this effect is mediated by Stn1, a target of NMD and a protein known to interact with the Pol12 sub-unit of Pol alpha. If so we will determine the molecular basis of the defect.

Finally we will screen to identify new genetic interactions that inform about the choreography of eukaryotic DNA replication.

Beyond academica we expect our work to impact several areas.

Synthetic genetic interactions affecting DNA replication in budding yeast identify potential drug targets that will be of interest to pharmaceutical companies. It is simplest to imagine two comparatively direct commercially relevant areas of relevance for our work. The first is anti-fungal drugs. Budding yeast is a fungus that shares many properties with other plant and human pathogens and therefore synthetic genetic interactions that reduce budding yeast cell fitness have the clear potential to be conserved in pathogenic fungi. If so, combination chemotherapy, using drugs that inhibit the same pair of targets we identify via budding yeast genetics, could provide a valuable way to prevent, treat or cure plant or animal fungal diseases. Since natural compounds (such as aphidicolin from the fungus Cephalosporium amphidicola, a broad specificity DNA polymerase inhibitor) target replication a single novel protein target may suffice. The second is in human or animal anticancer treatment. DNA and DNA replication are common targets for many anti-cancer regimes (radio and chemo-therapy). The insights we obtain into the role of specific proteins in coordinating DNA replication may provide novel insights (novel synthetic lethal interactions) identifying new ways to treat cancer.

Although we plan to perform experiments using yeast as a model organism but we will also do our best to make the implications of our discoveries clear to those working in other areas. We will encourage exploitation of our research by publicising our work at international meetings, national meetings and at the North East Fungal Forum (NEFF, http://research.ncl.ac.uk/neff). This open meeting is held every three months or so and features presentations by students, post docs, group leaders, and sometimes external speakers. NEFF is organised by post docs and students in the various fungal labs in the North East. Dr Marion Dubarry, the RA named on this project participates actively in NEFF.

NEFF meetings are advertised on the web in advance and flyers are posted around Newcastle and Durham universities and hospital. By advertising in the local hospital, the Royal Victoria Infirmary, we have engaged with clinical scientists involved in the treatment of human fungal diseases. NEFF is sponsored by a number of companies, in particular by a major British microbiology company, Formedium.

Finally DL and others in the lab are enjoy explaining their approach to local school children via Leading Edge (https://blogs.ncl.ac.uk/leadingedge). Each year we run a small practical project with a number of 13/14 year old children from a local school.