A molecular understanding of how MCM2-7 becomes loaded onto DNA to maintain genomic stability

Lead Research Organisation: Imperial College London
Department Name: Institute of Clinical Sciences

Abstract

Our cells contains DNA, representing a "genetic blueprint of life". DNA is composed of two complementary strands, which contain the genetic information in their centre. It is imperative that prior to cell division the DNA becomes duplicated, so that each daughter cell receives a full genetic complement from the mother cell. DNA duplication is a careful choreographed process, where numerous proteins synergize to assemble large DNA replication machinery at replication origins. Central for DNA duplication is an enzyme - the DNA helicase - that separates the two strands of DNA, to give copying machine access to the information that lies in the centre of the DNA. Initially the helicase must be loaded onto DNA, before it can become active and participate in DNA duplication. However, the helicase loading reaction is frequently misregulated during aging, cellular stress or in cancer, resulting in human disease or reduced crop yields in plants.
Fortunately, cells have evolved a highly efficient mechanisms that allows the loading of the helicase onto DNA. While this process in simple bacteria is well understood, our recent work has revealed that in eukaryotes this process is quite different. We have now uncovered evidence that the helicase itself is crucial for the loading process, which has not been seen to such an extent in bacteria. Now we want to uncover exactly how the helicase assembles on DNA and how it is loaded onto DNA, producing a ring shaped complex that encircles DNA.
We have created a number of helicase variants that "freeze" the helicase loading reaction at specific points. Additionally, we have developed several unique tools to analyse the detailed structural and functional changes associated with helicase loading. Therefore we propose for this programme to use these helicase variants to uncover how this molecular machine is able to be loaded in an accurate and processive way onto DNA and to define the organismal consequences of introducing the helicase variants in yeast cells.
Our work will generate a mechanistic framework, showing how helicase loading actually works. This will support the work of researchers in related fields, but could also lead to the development of helicase loading inhibitors with anti-fungal activity.

Technical Summary

The precise duplication of chromosomal DNA is essential for preserving the genetic complement of the cell. During the first step of DNA replication, termed pre-replication complex (pre-RC) formation, the replicative MCM2-7 helicase is loaded onto double-stranded DNA. Efficient and regulated loading of the replicative helicase is vital to maintain genomic stability and to prevent premature aging, growth defects in plants and tumorigenesis. Inhibition of helicase loading can occur due to mutations in helicase loading factors, inappropriate DNA secondary structures or due to altered expression of inhibitory factors, while excessive amounts of helicase loading proteins are associated with DNA re-replication, inappropriate homologous recombination and general genomic instability. Although we have identified the essential factors for helicase loading the overall process is only poorly understood, and the crucial mechanisms underlying efficient helicase loading and complex assembly remain to be established.
The aim of the proposal is to address how the replicative is loaded in a processive manner around DNA. We will be using an array of biochemical, electron microscopy and in vivo approaches to uncover the underlying mechanisms of helicase loading, a central process in DNA replication. The use of novel helicase mutants that promote specific arrests during the multi-step helicase loading process, will be used in combination with sophisticated biochemical assays and structural approaches to uncover crucial aspects of the helicase loading process. Together these studies will generate a mechanistic framework, which will allow us to better understand how altered helicase loading results in disease, aging or reduced crop yields. Moreover, we will test if it is possible to inhibit helicase loading, which could lead to the future development of inhibitors with anti-fungal activity that may improve human health and well-being in the long-term.

Planned Impact

The academic sector will benefit largely from the proposed project, through knowledge gain, development of new methods and technologies. The structural characterisation of large protein complexes by the PDRA in collaboration with Xiaodong Zhang (ICL) and Huilin Li (Brookhaven National Laboratory, NY, USA) will transfer some of this knowledge and technology to the group of CS and other associated groups - leading to enhanced research capacities. Application of this cutting edge technology will have positive impact on the DNA replication field and associated research areas, as it will allow the formulation of new research questions and will generate new results. The project will offer opportunity for career development and training, which will be readily transferrable to other related fields across the spectrum of Molecular Biology and Biochemistry. Indeed previous group members have been readily employed at research institutes, universities and in industry.
This project could have both short- and long-term socio-economic impacts for the United Kingdom. As with every research project the impact depends on the results obtained and is therefore difficult to predict. In the medium this project may spur the development of helicase loading inhibitors in yeast, thereby addressing a potential way to treat fungal infections. This could spur the commercialisation of scientific knowledge and products in the long-term. Related drugs that inhibit the loading of the human replicative helicase could generate new anti-cancer agents, which would benefit the UK. Importantly, the project would lead to employment of a PDRA, thereby contributing to the national economy. The interdisciplinary nature of the project will greatly enhance the training of the PDRA in high-end modern techniques ranging from biochemical, genetic to electron-microscopy based methods. Statistical analysis and modelling of structural and biochemical data will feature in the proposed research and the integration of the data from various data sets will demonstrate the power of this interdisciplinary approach. Such a trained PDRA (and associated PhD, technician, masters and undergraduate students) are likely to benefit UK biotechnology and pharmaceutical companies, as well as the university sector at national and international level. Finally, the general public will benefit from this work by becoming informed about our science in general and the project specifically. Indeed, we have planned a number of methods to engage with the public in different ways and to make our work as accessible as possible.

Publications

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Mendes ML (2019) An integrated workflow for crosslinking mass spectrometry. in Molecular systems biology

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Noguchi Y (2017) Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model. in Proceedings of the National Academy of Sciences of the United States of America

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Speck C (2016) Exceeding the limits - Cdc45 overexpression turns bad. in Cell cycle (Georgetown, Tex.)

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Tognetti S (2016) Replicating repetitive DNA. in Nature cell biology

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Yuan Z (2017) Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1. in Nature structural & molecular biology

 
Title 3D printed structures 
Description We have used 3D printers to print the structure of our newly discovered complex, was consequently painted and is now permanently displayed within the institute. 
Type Of Art Artefact (including digital) 
Year Produced 2018 
Impact We use this artefact during school visits to explain how molecular machines work. 
 
Description Identification of a protein structure, which shows in near atomic detail how the helicase loader engages the replicative helicase and highlights Cdt1 as a crucial factor in generating a topological link between the helicase and origin DNA. This work has already inspired us and others to explore the function of eukaryotic helicase loading in much more detail. This is important as helicase loading is a crucial mechanism for genome stability, which is important for healthy aging. Moreover, we starting to use the knowledge to develop inhibitors of helicase loading, which will be useful in the context of anti-fungicides and as chemotherapy.
Exploitation Route The data will serve as a rich resource for us to elucidate novel mechanism in DNA replication.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description A London consortium to establish a high resolution cryo-electron microscopy facility for research and training
Amount £3,000,000 (GBP)
Funding ID 206175/Z/17/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2017 
End 02/2022
 
Description A modern cryo-EM facility at Imperial College London
Amount £197,672 (GBP)
Funding ID 212938/Z/18/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2018 
End 09/2023
 
Description DFG Fellowship Max Reuter
Amount £72,000 (GBP)
Organisation German Research Foundation 
Sector Public
Country Germany
Start 11/2017 
End 10/2019
 
Description DFG Fellowship Sarah Schneider
Amount £72,000 (GBP)
Organisation German Research Foundation 
Sector Public
Country Germany
Start 07/2018 
End 06/2020
 
Description EPSRC Chris Weekes
Amount £130,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 10/2017 
End 09/2021
 
Description JSPS Fellowship
Amount £80,000 (GBP)
Organisation Japan Society for the Promotion of Science (JSPS) 
Sector Learned Society
Country Japan
Start 01/2019 
End 12/2021
 
Description Bruce Stillman 
Organisation Cold Spring Harbor Laboratory (CSHL)
Country United States 
Sector Charity/Non Profit 
PI Contribution Work on the biochemical and structural characterisation of replication complexes.
Collaborator Contribution Intellectual support and development of assays
Impact PMID: 19910535, 18647841, 28191893, 26305410, 25319829, 25085418, 23974098, 23851460, 22405012
 
Description Huilin Li 
Organisation Van Andel Institute
Country United States 
Sector Private 
PI Contribution We have prepared samples and helped with the interpretation of the results
Collaborator Contribution Electron microsopy sample preparation and analysis
Impact 19910535, 18647841, 28191893, 26305410, 25319829, 25085418, 23974098, 23851460, 22405012
Start Year 2006
 
Description Juri 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Preparation of crosslinked protein complexes
Collaborator Contribution Mass-spec analysis of crosslinked protein complexes
Impact Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1. Yuan Z, Riera A, Bai L, Sun J, Nandi S, Spanos C, Chen ZA, Barbon M, Rappsilber J, Stillman B, Speck C, Li H. Nat Struct Mol Biol. 2017 Mar;24(3):316-324. doi: 10.1038/nsmb.3372. Multidisciplinary: Biophysics, Chemistry, Biochemistry, Structural BiologyPMID: 28191893
Start Year 2015
 
Description Microscopy workshop for local primary school 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact We had 40+ school children visiting for a microscopy workshop hold at the MRC-CSC/Imperial College London, which engaged the children in biological questions and the school has asked for return visits due to its positive effect on science related subjects.
Year(s) Of Engagement Activity 2015,2016
 
Description School Visit (Grenfell Tower) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact We had students from a local high school visiting. The students came from the school near Grenfell Tower and were directly affected by the disaster. The workshop, including multiple presentations from my team, was very well received (including presentation on Noble Price winning technology and use of 3D printers to illustrate the technology) and the school reported increased interest in these subjects.
Year(s) Of Engagement Activity 2017
URL https://lms.mrc.ac.uk/lms-taster-sixth-form-science-students/
 
Description School visit (London) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I presented to a local school career information including a demonstration and practical training
Year(s) Of Engagement Activity 2019