The role of a novel family of eukaryotic DNA polymerases in mitochondrial DNA replication

Our cells contain DNA, the so called 'genetic blueprint of life' which encodes the information for our genes. DNA has a simple repeating structure composed of two complementary strands of DNA, which form a double-helix structure. This structure is contained in nearly every cell and must be copied as cells divide. Our cells produce a large number of proteins responsible for 'photocopying' this DNA in a process called DNA replication and it does this task with great accuracy. However, if this process goes wrong it can lead to the development of diseases, such as cancer. DNA polymerases are protein 'machines' that read the message of the 'parent' DNA and copy this to produce a new daughter DNA. We have discovered a new family of polymerases in human cells whose function is currently unknown. The focus of this work is to understand if these newly discovered proteins also have roles in replicating and repairing DNA and, if so, what specific role do they play in DNA replication.

The human genome encodes a variety of DNA polymerases required to replicate and repair DNA. Primases represent a specific class of polymerase that can synthesis short stretches of RNA (primers) at replication forks, which act to prime DNA extension by the replication polymerases. Only one DNA primase (Prim 1) has been identified and characterised in eukaryotes, which is required for the initiation DNA synthesis during replication. We have identified a second DNA primase family in higher eukaryotes and shown that these enzymes can synthesize RNA primers and also extend DNA substrates in vitro. This is the first example of a eukaryotic polymerase possessing both of these combined activities, making it unique in the eukarya. In addition, we present evidence to show that this novel primase functions in the mitochondria (mt), where it is involved in replicating the small circular DNA. Mt DNA encodes proteins essential for oxidative phosphorylation and exists as multiple copies in most cells. The exact mechanism of mt DNA replication remains to be established. The primary objectives of this proposal are to characterise the molecular and cellular roles of a novel primase involved in mitochondrial DNA replication in higher eukaryotic cells. We will employ biochemical, cellular and genetic approaches to elucidate the functions of these novel polymerases, both in vivo and in vitro. This research programme will provide valuable insights into the specific function of these enzymes in key DNA maintenance pathways (replication and repair) in eukaryotic mitochondria and advance our knowledge of the underlying machinery that replicates mt DNA in cells. Mutations in mt DNA are associated with serious human diseases. This research will also identify if mitochondrial primase-specific defects contribute to any of these aberrant cellular processes.

This proposal centers around the fundamental understanding of DNA replication processes and will have significant impact for research progress in this area, as well as contributing to the understanding of diseases related to DNA replication defects in mitochondria, such as cancer. In addition, understanding of basic DNA replication mechanisms, specific to mitochondria, will have significant impact on the development of novel biotechnology reagents and lead compounds for drug development that could open up exciting new leads for drug targets to treat cancer and mitochondrial syndromes. This research will help to train outstanding young scientists who will hopefully go on to set up their own research groups. These postdoctoral fellows in turn help to train undergraduate and PhD students. We have previously patented and developed a DNA repair system, developed from BBSRC-funded research, for application in the biotechnology sector. We will also patent this newly discovered enzyme, which has excellent potential for commercial exploitation for use in biotechnology and its development could lead to uses in cloning and related molecular biology kits. Notably, this system may also be an excellent drug target and the knowledge gained in our work will enable the rational design of small molecular inhibitors that could potentially act as lead compounds for future drug development. In order to showcase our research programme and maximise the impact on the broader industrial community, the University of Sussex, currently has two LTN business fellows, as well as a programme known as Curious, both programs designed to communicate academic research to the industrial sector. Curious is designed to communicate the research knowledge from within the University to the wider business sector. The aim of the event was to forge partnerships with external organisations, leading to knowledge exchange and collaborations and was developed in collaboration with South East England Development agency (SEEDA). In addition, the Research and Regional development office at the University of Sussex, regularly hold interactive events with the space known as InQbate. Recently, an event was held in relation to 'Aim Higher' to introduce Science, technology and maths research to young people in higher education. The University of Sussex has recently set up an on-line repository for all scientific and other research produced at the university, which is fully 'open access'. This means that our research will be deposited as electronic PDF files in this virtual 'library' for all to read, usually when they have been peer reviewed and accepted for publication. Much of our research output is produced by graduate students and their work is also deposited, in thesis form, to the British Library upon completion of their doctoral work. These results are also fully accessible to the public all around the globe. We will ensure maximum impact of our research by communication and liaising with the industrial and business sector, as well as with the public as described above. Support will be provided for this by the Regional Research and Development office, the IP office and the Enterprise office here at Sussex. New opportunities with business will be supported by the LTN business fellows. The interaction with the public and with schools will also be provided by the postdoctoral fellows.