Structure of origin DNA melting and unwinding complexes of a viral replication protein

The papillomaviruses are an important class of disease causing organism in animals and man. Bovine papillomavirus (BPV-1) is the model of the group and the consequences of disease (interference with suckling, milking and breading), has a significant economic impact worldwide. The highly related human viruses (HPV) cause cancer and therefore also have a negative impact on quality of life. The mechanisms of viral replication are consequently of significant interest as the viral replication proteins are important therapeutic targets. The survival and continuity of an organism, be it a virus or man, requires duplication of its genetic material- its DNA- and this is performed by specialized proteins. The first important step in this process is the recognition of specific 'origins of DNA replication' by proteins that separate, or "melt", the two otherwise paired strands of the DNA ('the DNA double helix'). In higher organisms like man, the timing and physical precision of this 'initiation' event is crucial to avoid the catastrophic consequences that may result from any loss of genetic material (for example, cancer). After the initial melting of the DNA at localized site, complete separation of the two helical DNA strands is performed by 'helicase' proteins. DNA strand separation by initiator and helicase proteins is generally poorly understood in simple organisms like viruses as well as higher species like man. However, the process appears to be highly conserved, suggesting that mechanism may be generally similar in all systems. We propose here to explore and characterize these replication processes in BPV-1. The advantage is that all the viral DNA replication activities are performed by a single protein called E1, thus simplifying the study. We will use powerful microscopes to take snapshots of different steps of the process and biochemical techniques that will help to define mechanisms. These studies will therefore contribute significantly to the understanding of the papillomavirus replication protein that is an important therapeutic target. Modeling these replication events in this simplified viral system will also help us understand the process in man. These studies will therefore assist in disease management and improving human and animal health.

The papillomaviruses are an important class of disease organism in animals and man. Hence, the mechanisms of viral DNA replication are of significant interest as the viral replication proteins are important therapeutic targets. The first steps in the initiation of double stranded DNA (dsDNA) replication are the specific recognition of the origin of replication (ori) followed by the localized melting of the dsDNA to expose un-stacked bases. In bovine papillomavirus (BPV-1), a discrete series of complexes of the initiator protein E1 have been characterized that assemble on ori to first melt and then unwind the DNA processively. The assembly series proceed from ori recognition complex (E1-ori, proposed to be a tetramer), to ori open complex (E1-oriOC, proposed to be a double trimer) and finally to replicative hexameric helicase (one or two assemblies). How each of these complexes engages their DNA substrates is unknown. In order to understand mechanisms it is essential to identify protein conformation changes that occur when substrates are engaged and the overall topological arrangement of the complex bound to their DNA substrates. Here we will to employ advanced cryo-electron microscopy, statistical analysis and image processing to obtain 3D structures of each protein-DNA complex. Crystallography has provided structures of several domains, which will help us to identify conformational changes of the complex. For the helicase complex, we will combine high-resolution biochemical technique for mapping DNA-protein interaction to obtain a detailed understanding of helicase-replication fork interactions. For the initiator complexes E1-ori and E1-oriOC we will rationalize new structural information with existing biochemical data to formulate mechanistic models. Given the similarities between E1 actions, those of its mammalian counterpart (MCM proteins) and the initiators of other model organism this study should reveal operating principals of universal applicability

Beneficiaries and interested parties:
(1) The Immediate beneficiaries include researchers in academia (national and international) and in the private commercial sector (pharmaceutical companies).
Interested academics are:
(i) Those in the immediate research area of viral/papillomavirus replication.
(ii) Those in the general research areas of DNA replication, helicase biochemistry and protein science.
(iii) Those who seek methodological advances in single particle analysis by electron microscopy.
(iv) Structural biologist employing diverse biophysical techniques to relate structure to function.

(2) Long-term direct beneficiaries would include:
(i) Veterinary scientists.
(ii) Those who rear cows, horses, mules or donkeys for economic use.
(iii) The wider population who will benefit from improved health and wealth that would accompany a reduction in papillomavirus disease.

The papillomavirus are important disease organism and the viral replication proteins are key therapeutic targets; this work could impact on drug development by the pharmaceutical industry. This remains a priority area even though current vaccines for HPV, that cause warts and cancer, are available. The latter are costly, of limited specificity, provide no benefits for those already infected and their long-term reliability is currently unknown. In the farm industry, BPV infection is of considerable economic importance. Teat papillomatosis can affect milk production and rearing of young animals. BPV infection also causes equine sarcoids in horses, donkeys and mules where genital infection interferes with breeding programs. This is particularly important in the third-world where there is a greater reliance on these animals for work. To the list of interested parties could also be added government policy makers who determine levels of overseas aid and also private third sector organizations, such as the Horserace Bet Levy Board, who seek to advance veterinary science and animal well-being.

Potential impact of the proposed work:
The work will advance our understanding of the mechanisms of DNA replication in a well-recognized and established model organism. Helicases are an important class of enzyme and they are therapeutic targets in cancer and viral diseases. They remain poorly understood. This is a protein structure-function study crucial for understanding these bio-molecules as therapeutic targets. Although incompletely understood, there is already significant structural and functional data for PV replication proteins that could facilitate a rational approach to drug design. The prospect that this and any new data emerging from our proposed study can be applied immediately is realistic. The nation's health and wealth would improve significantly if the disease burden of papillomavirus were alleviated in animals and man. Anti-papillomavirus drugs would have a direct impact on national health and reduce the financial burden on public health resources. Similar arguments apply to the cattle, dairy and equine industries whose commercial viabilities are enhanced when disease-free. Pharmaceutical companies that develop anti-viral drugs would derive wealth directly from their commercial products. Many of these have a significant research, development and production base in the UK. There is also the potential for patentable results as assays for screening therapeutic agents, for example small peptide inhibitors that target E1 replication activities could evolve from these studies. There will also be benefits from the continued training of postdoctoral research fellows and the development of their profession skills and creativity 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 also transferable between employment sectors.