Opening of a double stranded DNA replication fork by a hexameric helicase

The inner workings of mammalian cells involve a number of interrelated, small, "nanomachines". Individual machines are usually composed of a number of proteins that work together to perform one of the cell's functions in life, and determine its ability to survive and replicate. Understanding how individual machines function is crucial because when these machines breakdown the consequences can be catastrophic. For example, some cancers can be attributed directly to the failure of machines that process the genetic information (DNA) of a cell. In addition, invading microorganisms such as viruses bring their own machines that subvert or destroy a cell as it is hijacked for the pathogen's own advantage. The identification and understanding of how these machines function can therefore be a starting point for combating such disease states.
The biosciences can inspire nanotechnology, which aims to develop small mechanical devices based on protein machines that can be harnessed to drive or modulate molecular processes from which we benefit. Significant examples are found in biotechnology, such as emerging nanopore DNA sequencing devices that can operate at the single molecule level. The precise understanding of cellular nanomachines at the atomic level is absolutely dependent on the availability of precise structural information. For large molecular assemblies this information comes from X-ray structural analysis and, for very large assemblies, it is vastly assisted by powerful electron microscopes that can image single particles. Alone, electron microscopy can guide our understanding, but for the most part lacks the resolving power to provide critical detail. E1, the protein that we will study, is from a large group of pathogens, the papillomaviruses that cause warts and cancer in animals and man. Papillomavirus-derived diseases are of significant health and economic importance. E1 is often referred to as a "motor protein" that forms a six membered (hexameric) ring-like assembly, a machine that unwinds the two strands of the DNA molecule during replication (reproduction) of viral DNA. It can be viewed as a small radial engine that can move along DNA and separate its two strands and as such is a prototypic nanomachine and important target for drugs that could inhibit viral replication and disease.
Here we aim to understand how the E1 machine unwinds DNA. The objectives are to carry out detailed X-ray structural studies on E1 to determine its overall structure and how it interacts with DNA. We intend to verify these results and test models of function with complementary biochemical and biophysical studies. As a leading tractable model, it has great promise for being the first hexameric DNA-unwinding machine that we fully understand. The potential applications of our work include formulating strategies to target E1 and related helicases through structure based drug design. They will help understand replication processes in our own cells that are more complex and challenging to define. There are potential applications in nanobiotechnology and in medicine. Consequently, we envision that our studies will have a wide impact, with health benefits and improved living and economic standards nationally and internationally.

How hexameric helicases unwind dsDNA is unknown but an "active" mechanism of action assumes that the enzyme makes functional interactions with the replication fork junction (RFJ) to induce dsDNA melting. Structures of the papillomavirus E1 helicase domain (E1HD, residues ~300-605) have provided models for ssDNA translocation. We have now acquired a low-resolution structure of an intact E1 complex by electron microscopy (EM) showing an interconnected network of tunnels and chambers in its N-terminus (residues ~1-300). We also have an X-ray structure of E1HD bound to a RFJ-DNA substrate showing splitting of the dsDNA at the entrance to the C-terminal helicase domain. Accordingly, we hypothesise that the E1 N-terminus forms conduits for ssDNA and dsDNA and that the junction with the E1HD represents an active sites for DNA melting. Consequently, the E1 helicase is an active base pair separation machine.
The objectives are: (i) to obtain high resolution X-ray structural information for the E1HD bound to a RFJ-like DNA substrate. (ii) To test mechanistic models of dsDNA melting by site directed mutagenesis in combination with assays for E1 DNA binding, oligomerisation, ATPase and helicase activity. (iii) To obtain X-ray structural information for the uncharacterised N-terminal domain of the protein, map E1 surfaces involved in domain-domain interactions by NMR and provide corroborating biochemical and biophysical data for structure and activity of protein assemblies incorporating the E1 N-terminus, with and without DNA. (iv) Integrate all available high-resolution structural information to generate an accurate structure of E1. We should then be able to understand all protein-protein and protein-DNA interactions and deduce a model for DNA unwinding. This could be tested further by site directed mutagenesis and will inform future experiments to probe catalytic events in detail using single molecule techniques and mechanistic enzymology.

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 X-ray crystallography. (iv) Structural biologist employing biophysical techniques to relate structure to function. (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 targeting viral and cellular helicases for therapeutic gain. (ii) Veterinary scientists. (iii) Those who rear cows, horses, mules or donkeys for economic use. (iv) The wider population who will benefit from improved health and wealth that would accompany a reduction in papillomavirus disease.

Papillomaviruses are important disease organisms and their replication proteins are key therapeutic targets. Even though vaccines are available to HPVs that cause cancer, viral chemotheraphy is particularly relevant for the HPVs that cause genital warts (low risk of progression to cervical cancer), which currently cost Western health agencies much more to treat than cervical cancer. In the farm industry, BPV infection causes teat papillomatosis that can affect milk production and rearing of young animals. BPV infection also causes equine sarcoids 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 and food. Although BPV vaccines are available there are similar issues as with HPV vaccines concerning the lack of protection against all serotypes and their unaffordability in developing nations. Although there is a drive in Western nations to reduce the drugs given to reared animals in the food chain, economic arguments make this harder to justify in developing nations. Interested parties may also include government policy makers who determine levels of overseas aid and third sector organizations, such as the Horserace Betting Levy Board (veterinary science and animal wellbeing).

Potential impact of the proposed work:
It will advance our understanding of the mechanisms of DNA replication in a key model system. Helicases are important therapeutic targets in cancer and viral diseases but remain poorly understood. This is a protein structure-function study and atomic structures coupled with functional data can facilitate a rational approach to drug design. The prospect that this and any new data emerging from our studies can be applied immediately is realistic. 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 from anti-viral products. Many pharma companies have a significant research, development and production base in the UK. There is also the potential for patentable results as small molecule inhibitors and assays for screening therapeutic agents could evolve from these studies.
There will also be benefits from the continued training of postdoctoral research fellows and the development of their professional 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.