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

Lead Research Organisation: Birkbeck College
Department Name: Biological Sciences

Abstract

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.

Technical Summary

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

Planned Impact

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.

Publications

10 25 50
 
Description • Significant new knowledge generated;
• New or improved research methods or skills developed;
• Important new research resources identified;
• Important new research questions opened up;
• Significant negative results and/or research paths closed off;
• Particularly noteworthy new research networks/collaborations/partnerships, or combinations of these;
• Increased research capability generated from training delivered in specialist skills;
• Summary information combining outcomes detailed in other sections.
Helicases are proteins that are essential for replicating DNA, the genetic information that encodes for life. DNA is composed of two strands that must be separated before it can be replicated and this is done by helicases. With Prof. E. Orlova (Lead applicant, Birkbeck College London) we have used high-resolution microscopy (electron microscopy, EM) to visualize for the first time a replication helicase from a pathogenic virus (papillomavirus) bound to the DNA that is being unwound- a DNA replication fork with a double-stranded and two single-stranded components. The papillomavirus helicase is a six-subunit ring-shaped complex. We showed that the helicase complex has an interconnected network of internal tunnels and chambers never previously observed, suggesting a mechanism for how DNA could be bound and unwound by the helicase. This information is important, as helicases are drug targets in viral replication and also cancer. An understanding of how they function is critical for developing therapies.
To further understand the DNA unwinding mechanism we developed a new method to map DNA interactions with the protein complex. We bound protein tags to the DNA ends that then allowed us to directly visualize their entry and exit points in the protein complex. We also improved available methods to sort and characterize the protein complexes observed by electron microscopy. This then allowed us to trace the path of all arms of the DNA replication fork through the helicase according to the observed tunnels, and hence map the position of the replication fork junction where DNA is splitting. Our results therefore represent a significant technical advance in the characterization of protein complex by EM as well as indicating how a replication helicase engages a DNA replication fork. With accompanying biochemical data, our results provide the first mechanistic insight into how hexameric helicases unwind DNA. Significantly, our data show that DNA strand separation occurs inside the helicase complex not externally by "steric exclusion" as previously assumed. The results also open up significant new research questions, such as how helicases can cope with obstacles such as other proteins that can be bound to DNA.
During the project the research capabilities of two postdoctoral researchers (PDRA) were extended as they learnt and developed new methodologies in structural biology and biochemistry. A PhD student involved in the project was also trained in these research methods. We extended and consolidated our links with Prof. A Antson's group in the University of York and developed new approaches to investigating helicase complexes with high resolution X-ray crystallography.
Our principal findings have been published as a peer reviewed article in a leading scientific journal. We anticipate subsequent publications detailing higher-resolution structural information to follow.
Exploitation Route Our findings have implications for understanding the action of helicases which are an important class of protein required for genome stability and the prevention of diseases such as cancer. Our structural data are for the replicative helicase of papillomavirus, an important group of human and animal pathogens. For the first time they demonstrate "active sites" in DNA unwinding in the N-terminus of the helicase complex that is critical for viral propagation. These sites could be the targets of chemical inhibitors and hence drug-like molecules that may progress to effective therapies for viral disease. Our findings are also significant for the understanding of protein nanomachines that are exploited by synthetic biologists. They hope to create robotic devices from proteins that generate movement.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The findings have been used for chemical to produce samples for electron microscopy, to fund two postdocs who are involved into experimental studies, to attend GRC conference and the Student retreat.
First Year Of Impact 2014
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description practical courses on electron microscopy
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact Providing teaching materials,, raising awareness of importance of safety in research, importance of biological sciences for medicine, public services and environmental sustainability
 
Description Collaborative Computational Project for Electron cryo-Microscopy (CCP-EM): Supporting the software infrastructure for cryoEM techniques. 2015- 2020.
Amount £1,177,000 (GBP)
Funding ID MR/N009614/1 
Organisation Medical Research Council (MRC) 
Department MRC Partnership Grant
Sector Academic/University
Country United Kingdom
Start  
 
Description State of the art cryo electron microscope for molecular and cellular biology, Cryo-electron microscopy
Amount £2,340,000 (GBP)
Funding ID 206166/Z/17/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2016 
End 12/2018
 
Title Analysis of heterogeneity of samples 
Description Statistical methods for separation of molecular images according to their sizes or conformations 
Type Of Material Biological samples 
Year Produced 2009 
Provided To Others? Yes  
Impact Usage of the new tool by researches in structural biology using EM 
 
Title EM structural data 
Description Structural densitiy maps of portal proteins 
Type Of Material Biological samples 
Provided To Others? Yes  
Impact Densty maps for the core complexes 
 
Title Electron microscopy 
Description Analysis of the T4S systems in different organisms. The T4S systems are involved into active pathogenesis of many bacteria. We study the structural bases of the complexes to reveal a mechanism of thier activity. 
Type Of Material Biological samples 
Provided To Others? No  
Impact Possible development of drugs, that would prevent pathogenic activity of bacteria and preventing of the horizontal transfer of genomic material between bacteria, a reason for their high resistance to antibiotics. 
 
Title Electron microscopy of biocomplexes using low and high end microscopes. Structural analysis of complexes form medium to high resolution. 
Description Firstly we are using cryo electron microscopy and automated data collection. Then we apply methods of image analyses such as determination of transfer function of the microscopes and statistical analysis of images, finally determination of orientations of particle images with three-dimensional reconstruction. That step is followed by interpretation of densities obtained during the reconstruction and analysis of variations in structures due to effects of the substrates. 
Type Of Material Biological samples 
Provided To Others? Yes  
Impact These methods allow us to improve a resolution of the EM structures and reveal conformational changes related to the function of the biocomplexes 
 
Title Flexible fitting of atomic structures 
Description Using Coot (X-ray software package), Phenix , and Em Flexible fit to make fitting of atomic models (both known X-ray structures and predicted based on homology modelling) . That allows us to make a reliable interpretation of bio complexes on the atomic level and to understand function-conformational changes that take place during proteins interactions 
Type Of Material Biological samples 
Year Produced 2014 
Provided To Others? Yes  
Impact The methods is not absolutely unique, but it was essential to understand the function of the E1 heleicase and T4SS 
 
Title Image processing of EM micrographs 
Description Structural analysis of biological samples (proteins and macro biological complexes using digital approaches for alignment of images, their classification, determination of space orientation of the images and finally using methods of structural reconstructions of bio complexes in space. 
Type Of Material Biological samples 
Provided To Others? Yes  
Impact Approach has been implementsed in the package IMAGIC and broadly used in electron microscopy/ 
 
Title EMDB 
Description This is a database (EMDB) that archives all structures obtained by electron microscopy (in negative stain and in cryo). All our results are deposited to this data base became available to other scientists 
Type Of Material Database/Collection of data 
Provided To Others? Yes  
Impact All our deposited data became available to the broad EM community and helps to analyse similarity and differences between bio complexes and investigate their functional activity. 
 
Title PDB 
Description The Protein Data Bank archive (PDB) serves as the single repository of information about the 3D structures of proteins, nucleic acids, and complex assemblies. It allows to validate structures and assess their quality using widely accepted standards and criteria. 
Type Of Material Database/Collection of data 
Year Produced 2008 
Provided To Others? Yes  
Impact That helps to us and other groups to make reliable interpretations of complexes and understand their functionality 
 
Description Analysis of helicases 
Organisation University of Massachusetts
Department University of Massachusetts Medical School
Country United States 
Sector Academic/University 
PI Contribution We have obtained the first structure of E1 helicase and reveled areas of interation with DNA. At the present moment we doing refinement of E1/DNA cmplexes and approaching sub-nanometer resolution. The manuscript is in preparation.
Collaborator Contribution This is a collaboration : the collaborators provide samples and do mutagenesis.
Impact This collaboration is multi-disciplinary: inludes electron microscopy, biochemistry, and image analysis. One manuscript has been published 26240379 and the next one is in preparation.
Start Year 2011
 
Description Structural analysis of bacteriophages 
Organisation Institute for Integrative Biology of the Cell (I2BC)
Country France 
Sector Academic/University 
PI Contribution During the last decade, we determined and published several EM structures of different components of the bacteriophage system: a portal protein, an isolated head-to-tail interface, a tail, and a capsid. We now report structures of the supramolecular complex forming the complete portal-tail interface extracted from functional phages in a form still able to react efficiently with the host receptor like in the infectious phage system . These nearly in vivo experiments combined with mutagenesis, structural EM analysis and modeling provide major novel functional and mechanistic insights on this structure.
Collaborator Contribution my collaborator Prof P. Tavares has very close interaction with my group and provides samples of purified complexes and performs mutagenesis to reveal a mechanism of DNA deliver to host cells, in another words a mechanism of infection by bacteriophages. At the present moment
Impact PMID: 22514336, 19433794, 17611601, 17363899, 12628918, 11501993, 10467096, 8890151, 26278173, 25991862 Viral Molecular Machines, Chapter 25, , ISBN 978-1-4614-0980-9 Orlova, Elena V. (2012) Bacteriophages and their structural organisation. In: Kurtboke, I. (ed.) Bacteriophages. Rijeka, Croatia: InTech, pp. 3-30. ISBN 9789535102724.
 
Description X-ray studies of small protein components 
Organisation University of York
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We have done sturctural studies of several proteins form SPP1 bacteriophage: capsid protein, portal protein and some tails protein at low resloution. Em densities were used to do phasing of X-ray data and to obtained the X-rail structure at a resoltuoin on 3A.
Collaborator Contribution In the studies of the E1 helicase we expect to collaborate with Prof. A. Antoson ion of the atomoc structures of small components of the origin complex to reveal the mechanism of prtein/DNA interactions
Impact We had a very productiov collaboration on studies of the Bacillus subtilis nacteriophage SPP1 and have published a structure of the portal protein in EMBO J, in 2007 (Structural framework for DNA translocation via the viral portal protein Lebedev, A. A., Krause, M. H., Isidro, A. L., Vagin, A. A., Orlova, E. V., Turner, J., Dodson, E. J., Tavares, P. & Antson, A. A. 4 Apr 2007).We aim to combine X-ray crystallogrpahy and cryo-electron microscopy to reveal dynamic of macromolecular complexes.
Start Year 2017
 
Description ISMB Retreat 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact More as 150 PhD students and posdocs from UCL and BIrkbeck college attended the retreat.
Typically it has very engaging conversation between students and senior researches related to the future plans and possible activities within and nearby sciences.


Broadedning the mind a vision for students , to be reflected in their future plans both in scince and close to scince areas, like publications, editoral jobs etc.
Year(s) Of Engagement Activity 2007,2009,2010,2013
 
Description EMBO cryo course on image processing 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Postdocs and PhD students were given lectures on principles of image analyses in electron microscopy. A number of world known scientists gave talks on their research and attendees had ana ample opportunity to talk to famous scantiest and to ask the about future plans and potential jobs.
Year(s) Of Engagement Activity 2015
 
Description GRC Conference , Three Dimensional Electron Microscopy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The open discussion related to the efficiency of the new equipment used in EM and practicality of new methods in the field of EM It has been discussed the efficiensy

After discussion the issues of impact on our research of new detectors has been assesced and new possible test were suggested.
Year(s) Of Engagement Activity 2012,2014
 
Description ISMB Retreat 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact More as 150 PhD students and posdocs from UCL and BIrkbeck college attend the retreat. Typically it has very engaging conversation between students and senior researches related to the future plans and possible activities within and nearby sciences.

Broadedning the mind a vision for students , to be reflected in their future plans both in scince and close to scince areas, like publications, editoral jobs etc.
Year(s) Of Engagement Activity 2008,2009,2011,2013,2014,2015
 
Description IUCr 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Organising the future 2017 congress on crystallography. Considering potential key note speakers and sessions related both to electron crystallography and high resolution microscopy. Organising sponsorship to support of speakers and students and postdocs who are involved in research related to the electron crystallography.

It was an active discussion between the scientists involved in studies of materials by methods of electron crystallography. As chair of the Committee Electron Crystallography of the IUCr it was important to explain how the methodology developed in material sciences and biology can be merged, shared and developed for the mutual progress during international conferences, workshops and schools organised by the members of CEC.
Year(s) Of Engagement Activity 2014,2015,2016,2017
 
Description Open days in university 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact 400 people attended the event. During the open days I have been engaged into inter5sting conversation related to explanation of what is it structural biology, its role in medicine and education, improvement of environment.

Increasieng a number of undegraduates and graduates in BIrkbeck College
Year(s) Of Engagement Activity Pre-2006,2006,2007,2010,2012,2013,2014,2015
 
Description Participation on Open days of Birkbeck college. 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Explanation of general ideas biolcogical studies, importance of structural studies and how it can be achieved. Making the links between microbiology, structural studies and development of means against diseases. Explanations of how the mutations in biological complexes can cause cancers and what we have to understand to be able to i=restore the functions of these molecules
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015,2016,2017,2018,2019