Structure and biochemical mechanism of DNA replication initiation machines
Lead Research Organisation:
Birkbeck, University of London
Department Name: Biological Sciences
Abstract
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Technical Summary
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.
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.
Planned Impact
Beneficiaries and interested parties:
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:
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
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:
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
People |
ORCID iD |
| Elena Orlova (Principal Investigator) |
Publications
Ignatiou A
(2019)
Structural transitions during the scaffolding-driven assembly of a viral capsid.
in Nature communications
Javed A
(2021)
Unwinding of a DNA replication fork by a hexameric viral helicase
in Nature Communications
| Description | We analyze structures of the DNA replication complexes from the papillomaviruses (PV), that cause warts and serious diseases such as cancer. We aim to reveal interactions between three components: two viral proteins, E1 and E2, and viral DNA that should be replicated. E1 and E2 make a complex that binds the DNA at a specific site that is named as an origin to start the replication. We have obtained the complexes of E1/ E2/ DNA and E1/DNA from Dr. Sanders (Sheffield University). A hexamer of E1 represents of a class of biological machines known as helicases that unwind the viral DNA prior its replication. Failures in helicase function cause serious diseases such as cancer and they are also important drug targets against microbial infections. Despite of importance of this information it is still unclear how hexameric helicase works. We want to understand how three protein-DNA complexes operate together during replication through structural analysis of different complexes (i) Having samples from from Dr. Sanders lab were optimized for the freezing conditions of complexes for the data collection on high-end microscope to obtain the structures at high resolution. For that we have checked samples initially using electron microscopy in negative stain and started to do analysis using cryo electron microscopy. However, screening of samples has indicated the complexes were not very stable preventing the efficient structural determination. So we have contacted Dr. Sanders and made a few suggestions both for the preparation of complexes and arrangements of their stability. At the current moment we have collected new data set and started its analysis. (ii) Samples of a complex comprising a hexamer of E1 and the viral DNA. The E1 hexamers engaged with a replication fork have a specific point where the two strands of DNA became separated. These complexes were screened on low-hand microscopes and from the best grids with good distribution of samples EM data were collected firstly on the local Polara microscope and later on Diamond EM facilities. Analysis of images has demonstrated that the complex is highly flexible especially in the area of DNA binding site. Dr. Sanders has screened a number of chemical reagents for their ability to cross-link and stabilize the protein complex and these samples will be analyzed using middle level microscopes. Our initial data demonstrated how the complex engages with a replication fork and the point of DNA unwinding. However, the further improvement of the complex stability will be helpful to allow us to improve the structures and to describe the reaction mechanism in details. (ii) We encountered similar problems (instability and aggregation of complexes during preparations for electron microscopy) with samples of the PV E1-ori complex that initiates the replication process. Dr. Sanders screened chemical agents for their ability to stabilize E1-ori complexes and facilitate the isolation of stable assemblies. He has also screened small co-factors for their ability to influence complex assembly. These samples were screened using low-hand mciroscopes in negative stain, and we have found that the samples became more stable and for the first time we have been able to obtain informative images where the particle images were consistently aligned and allowed to improve signal/noise ratio reveling characteristic features of the complex. Our new findings using Cryo_EM revealed heterogeneity in the complexes of E1-ORI that interacts with DNA. The hexamers of E1 helicase have significant distortion in symmetry due to interactions with DNA |
| Exploitation Route | Our findings will inform researchers on improved ways to proceed with their own research or develop new ideas to test. The E1 helicase is a small "nanomachine". Our work could also have a significant impact in synthetic biology that utilizes or exploits molecular machines based on cellular systems. In the case of E1 this could be direct, or through the exploitation or adaptation of its operating principles that we can now better understand |
| Sectors | Chemicals,Education,Healthcare,Pharmaceuticals and Medical Biotechnology |
| Title | Electron microscopy of biocomplexes using low and high end microscopes. Structural analysis of complexes form medium to high resolution. Bioinformatics |
| 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. using tools for analysis interactions between proteins and DNA such as COOT, PISA and sites on CCP4 |
| 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 and undestnad functionality of the complex |
| Title | The E1RF map and atomic model are deposited to the EMDB data base under accession codes EMD-11852 and 7APD [https://doi.org/10.2210/pdb7APD/pdb] correspondingly. Source data are provided with this paper. |
| Description | Purified E1RF complex at ~0.05 mg/ml were applied to lacey carbon grids with a continuous carbon support film (EM Sciences) and vitrified using a Vitrobot Mark IV (ThermoFisher TM). Data for the E1RF complex were collected using EPU software (ThermoFisher TM) on a Titan Krios electron microscope (ThermoFisherTM) operating at 300 kV and equipped with K3 Summit direct electron detector (Gatan Inc.) at the eBIC Diamond light source facility (Harwell, Oxfordshire, UK) and Birkbeck College, 11,200 movies were collected ans processed. For particle picking crYOLO v1.3.6 50 has been used. The extracted particle images were then subjected to two-dimensional (2D) classification in RELION 3.0 and the subset of the images that comprised the best classes, showing secondary structural features, was exported subsequently to cryoSPARC v2.9.0 31. All following steps in image processing were carried out in cryoSPARC. CryoSPARC, using the 3D map of the E1RF complex obtained during the first step of 3D classification. The final 3D map was obtained at a resolution of 3.89 A° at 0.143 FSC threshold (and 4.5 A° at 0.5 FSC threshold). For the fitting, the map was sharpened using option Auto-Sharpen in PHENIX v1.14 51. Fitting into the final cryo-EM E1RF map was done using as a starting model the X-ray structure of the E1 helicase domain with ssDNA and bound ADP (PDB 2GXA), |
| Type Of Material | Data handling & control |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | Our data are in full accord with previous structural models , biochemical data, and smFRET observations . The E1 protein participates in the replication process, using both the E1HD and OBD domains for dsDNA ori binding, melting37,38,45 and processive DNA unwinding. PV E1 demonstrates how viruses have borrowed functional segments from eukaryotic cells (e.g. the AAA+ domain) and have mimicked the operating principles of the host cell replication initiation apparatus (e.g. the CMG/fork protection complex) to generate a minimalistic but highly streamlined replication machine. Understanding of these viral proteins will help to improve our knowledge of the more complex cellular replication machines and how viruses could be targeted therapeutically when they emerge as threats. |
| Description | Analysis of of mutation effects in the helicase activity through structural studies |
| Organisation | University of Sheffield |
| Department | Sheffield Medical School |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Structural analysis provided possible points of mutations that can affect activity of the complex and explain principles of the DNA replication |
| Collaborator Contribution | Biochemical experiments checking consistency between suggested hypothesis and observed activity of the complex in study |
| Impact | The studies are in the stage of verification |
| Start Year | 2018 |
| Description | EM study of E1 replication complexes of papilloma virus |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Postgraduate students |
| Results and Impact | Description of methods of electron microcopy in the analysis if biologically active complexes, that is reflected in different molecular conformation. Explanation of basic structural methods that were used in the analysis of the E1 helicase. |
| Year(s) Of Engagement Activity | 2022 |
| 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 |