Elucidating the molecular basis for DNA primer synthesis

Our cells and those from all living organisms contain DNA, the so called "genetic blueprint of life", that encodes the information for all our genes. DNA has a simple repeating polymeric structure composed of two complementary strands of DNA composed of bases, which form long, string-like, double-helical structures that make up the genome. Our genome is packaged away into chromosomes, contained within the nucleus of nearly every cell. This information must be faithfully copied as cells divide to produce daughter cells. Cells produce a large number of proteins responsible for "photocopying" this DNA blueprint. The proteins tasked with accurately copying the cell's genetic code are called DNA polymerases. However, polymerases are unable to produce or copy DNA from scratch and require the synthesis of a short primer from which to copy the bulk of the genomic DNA. Fortunately, all cells possess a priming enzyme called DNA primases, whose role it is to make these initiating primers, although how they produce these short polymers from is still poorly understood.

In this research programme, we are proposing to determine how DNA primases, from bacteria and human cells, are able to perform the essential task of primer synthesis. This proposal will provide critical insights into this fundamental replicative mechanism that is essentially required for DNA synthesis in all cells. Understanding priming mechanisms that initiate and maintain efficient DNA replication will help uncover new strategies to treat conditions and diseases associated with uncontrolled proliferation, such as cancer and microbial infections.

Replicative DNA polymerases are exquisitely fine-tuned to synthesise DNA in a highly accurate and efficient manner however, they are unable to initiate DNA synthesis de novo. As a consequence, DNA replication requires an initiating step to generate a primer containing that is requisite for polymerase-dependent synthesis. Early studies identified that this 'priming' role is fulfilled by specialized polymerases called DNA primases capable of synthesising short primers that enable DNA synthesis. However, the activities and cellular roles of these enzymes extends further than this essential role in initiating DNA replication. Over recent years, significant evidence has accumulated establishing that primases undertake a wide variety of cellular roles including replication, repair, and damage tolerance. Although Prim-Pols and DNA polymerases share a common metal-dependent mechanism of polymerization, much less is known about the initiation step of de novo primer synthesis.

The major aims of this proposal are to elucidate the molecular mechanisms employed by two distinctive Prim-Pols involved in primer synthesis from both bacteria and eukaryotes. We will use a variety of biochemical, biophysical and structural approaches to provide complementary mechanistic insights into the modus operandi of these important replicative enzymes to delineate how these proteins initiate de novo primer synthesis and extension, which is requisite for all cellular life. Together, these studies will significantly enhance our understanding of how Prim-Pols initiate DNA synthesis, one of the most important processes in cells. These studies are also likely to provide critical insights into how these processes might be targeted by novel inhibitors and how these pathways may also contribute to disease.