Structure and biochemical mechanism of DNA replication initiation machines

Proteins are essential components of cells that do all the jobs that ensure the continuity of life. Some of them assemble to form "nanomachines" that do work. This work often involves rearranging or remodelling small molecules, other proteins or the DNA that encodes the information of life. Some of these machines are made up from groups of proteins that work together but others are proteins that act alone. Understanding how individual biological machines function is crucial because if they fail the consequences can be catastrophic for living organisms. For example, some cancers and neurodegenerative disorders are caused by the failure of nanomachines that process the DNA in our cells. In addition, pathogens that invade the body, such as viruses, bring their own nanomachines that destroy healthy cells or hijack them to make the virus stronger. Identifying these machines and understanding how they function can therefore be a starting point for combating diseases.
We are studying the DNA replication proteins from a group of pathogenic viruses, the papillomaviruses (PV), that cause warts and serious diseases such as cancer. Accurate replication of our own DNA is essential for genome stability and healthy ageing. These systems have many components and to understand the process we require knowledge of the underlying mechanisms. The viral proteins we are studying, known simply as E1 and E2, bind to DNA and change its structure so that it can be replicated. In our own cells a complex assembly of many different proteins is required to do the same jobs that the PV E1 protein does. The aim of our research is to determine how the viral proteins E1 and E2 function on DNA during replication, to understand processes that are essential for life where potential therapeutic strategies could emerge to improve health and well-being.
To do this we have designed ways to characterize these exceptionally small protein machines. Just as complex man-made mechanical machines could never be understood without understanding how they are made up, we need to know the structure of protein nanomachines to know how they work. We use biochemistry, powerful electron microscopes and computer technology to generate images of protein complexes and then use this information to re-construct their detailed three-dimensional structures. In parallel we apply biochemical techniques to understand the reactions performed by these proteins and relate this information to the structures we observe. Ultimately, we expect that our studies will have a significant impact in the healthcare sector and wider economy. The potential applications of our work include designing drugs to target viral proteins required for their replication.
This research is important since it is estimated that up to 5% of all cancers are caused by human papillomavirus (HPV) and up to 600 million people are infected with HPV at any one time but there are currently no drug treatments of proven efficacy. Our studies will also inform us of how the more complex cellular counterparts of the PV replication proteins work, where there are further therapeutic applications to treat cellular and microbial (e.g. fungal) diseases. There are also potential applications in nanobiotechnology. This branch of science aims to develop small mechanical devices based on protein machines that can be harnessed to drive or alter molecular processes for our benefit.

How pathogenic papillomaviruses (PV), which replicate in mammalian cells, initiate DNA replication mimics that of their host. Mammalian DNA replication is tightly regulated to maintain genome stability and prevent serious degenerative disorders like cancer. The activation of an origin of DNA replication (ori) involves the ATP-dependent assembly of oligomeric initiation complexes. Hexameric helicases assemble at replication forks after ori recognition and localized double-stranded DNA (dsDNA) melting. So far no structural information is available for prokaryotic or eukaryotic initiator complexes that describe clearly how dsDNA is remodeled during replication initiation. Using electron microscopy (EM) and single-particle analysis (SPA) we have acquired images and structures of PV replication pre-initiation, initiation and helicase complexes bound to DNA. Our aim is to determine how PV initiator complexes assemble and drive the DNA structural changes required to establish a replication fork.
The objectives are: (i) to determine by cryo-EM and SPA the structure of a PV replication pre-initiation complex composed of the initiator protein E1, transcription factor E2 and ori DNA. Understand the ATPase switch in E1E2-ori that is required to establish a replication initiation complex. (ii) to obtain cryo-EM structures of E1-ori initiation complexes to deduce the mechanisms of ori melting and helicase assembly. (iii) to obtain a high-resolution cryo-EM structure of the hexameric E1 helicases bound to a replication fork-like DNA substrate, revealing all protein-DNA interactions involved in dsDNA unwinding. Mechanistic models for dsDNA processing will be probed by generating variant E1 proteins by site directed mutagenesis for testing using biochemical and cell-based replication assays. Consequently, we will develop a framework to understand cellular DNA replication initiation and viral initiation as a target for anti-viral drugs.

Beneficiaries and interested parties: who might benefit from the proposed research?
(1) The immediate beneficiaries are national and international researchers in academia and industry including those (i) investigating DNA replication initiation complex and helicase structure and function; (ii) in the general fields of DNA replication, genome stability and protein-nucleic acid interactions; (iii) who seek methodological advances in structure determination by electron microscopy and single-particle analysis; (iv) structural biologists employing biochemical and biophysical techniques to relate structure to function; and (v) researchers in bionanoscience who are developing synthetic molecular machines based on cellular systems.
(2) Long-term direct and indirect beneficiaries would include: (i) researchers in pharmaceutical companies (e.g. medicinal chemists) targeting PV DNA replication machinery, helicases and related cellular enzymes for therapeutic gain in humans and animals. (ii) The wider population who will benefit from improved healthy life expectancy and reduced healthcare costs that could accompany new therapeutic approaches.
Potential impact of the proposed work: How will the interested parties benefit from the proposed research?
Researchers who study the structure and function of DNA replication machines will benefit because we will share information from our tractable system that is able to reveal the molecular events associated with DNA processing during eukaryotic replication initiation at unprecedented levels of detail. New mechanistic insights will serve as models for understanding related nucleic acid processing enzymes, extending the impact to a broader group of researchers in the biological sciences.
Helicases are critical enzymes for genome stability and defects are associated with serious degenerative disorders. The PV replication protein E1 is a potential therapeutic target for human and animal infections. BPV causes severe infections (e.g. teat papillomatosis) that are particularly problematic in developing nations. HPV causes common warts but also serious sexually transmitted diseases leading to cancer and it is estimated that up to 600 million people world-wide are infected. However, no effective virus-specific therapies are available. We will provide a detailed understanding of mechanisms required to formulate rational approaches to drug discovery. Many pharma companies have a significant research, development and production base in the UK so there is the potential for significant economic benefit.
Researchers in the bionanosciences will benefit because we will use the E1 replication protein to describe in detail how this class of bionanomachine works. This information could be exploited to develop synthetic machines that improve human and animal health. Helicases are employed in nanopore sequencing devices that can read tens of kilobases of sequence in real time at low cost. They have many applications, including rapid diagnosis of infections and continuous health monitoring, but there is a need for more efficient machines.
Structural biologists, computational scientists who develop methods for single-particle analysis and protein scientists in general will benefit. The Institute of Structural and Molecular Biology, London, hosts cutting-edge research groups including Prof. E. Orlova who is developing methods for structure determination by electron microscopy that are used by the global research community.
The project will provide an opportunity to train PDRAs in cutting-edge methods for the analysis of protein structure and function. They will develop additional professional skills and creative ability that could be integrated into any commercial or academic enterprise requiring a highly skilled structural biologist or protein biochemist. Many of the skills that will develop, such as time management, team working, communication and technical, are transferable between employment sectors.