Characterisation of Mcm10 protein as a target for development of new anticancer drugs.

Lead Research Organisation: University College London
Department Name: The Wolfson Inst for Biomedical Research

Abstract

The protein machinery controlling DNA replication is a major regulator of cell growth in human tissues and is frequently deregulated in cancer. We are trying to understand how different parts of this protein machinery work together.
Our work is focused on a conserved protein called Mcm10, which connects parts of the DNA replication machinery and is vital for the DNA copying process. We have recently solved the molecular structure of human Mcm10. The structure is a ring formed by six Mcm10 molecules. This gives us a very good idea of how this important protein may connect parts of the DNA copy machinery and DNA at the same time. The structure and a powerful multidisciplinary approach give us an excellent starting platform for molecular and cell biology studies of Mcm10.
Recent advances show that proteins, which form DNA replication machinery, are attractive targets for the development of new treatments against human disease such as cancer. Understanding the Mcm10’s molecular blueprint and its role in DNA copying process will help in designing novel therapeutic agents, which will selectively target and eliminate cancer cells without damaging normal healthy cells.

Technical Summary

The protein machinery controlling DNA replication is a major regulator of cell growth in human tissues and is frequently deregulated in cancer. Initiation of DNA replication is tightly regulated and starts with the ordered assembley of origin recognition complex (ORC), Cdc6, Cdt1 and Mcm2-7 proteins into pre-replicative complexes (preRCs). These preRCs are activated by CDKs and Cdc7/Dbf4 kinase and the loading of Mcm10 protein. The latter event triggers the transition from preRC to pre-initiation complex (preIC) which is followed by the recruitment of Cdc45 and the DNA polymerase-a machinery.
Emerging data point to the existence of a DNA replication initiation checkpoint which monitors the assembly of a critical number of preRCs. When initiation of DNA replication is blocked it results in an apoptotic response in cancer cells and reversible cell cycle arrest in normal cells. This makes preRC formation and the transition to the preIC a promising target for anticancer therapies capable of blocking cellular proliferation without causing genotoxic stress.
The Mcm10 DNA replication initiation factor is a conserved nuclear protein critical for origin activation. During the preRC to preIC transition Mcm10 is vital for origin unwinding and provides a link between the Mcm2-7 replicative helicase and DNA polymerases. The properties of Mcm10 as an integral part of the replication machinery initiation make it an important target for therapeutic intervention.
We have recently solved the first structure of human Mcm10 by using electron microscopy and image analysis. The Mcm10 molecule is a ring-shaped hexamer. The structure suggests a model of how this protein may serve as a connector between the Mcm2-7 helicase and DNA polymerase. The ring-shaped molecule may serve as a docking platform for assembly of the molecular machinery required for initiation of DNA replication. The structure allows us to predict the areas of DNA binding, major domain boundaries and positions of Zn-fingers potentially involved in DNA binding and/or protein-protein interaction.
This model gives us the structural background to start molecular and cell biology studies of the Mcm10 structure-function relationship. We now aim to obtain detailed information on functional domains of Mcm10 involved in DNA-binding and protein-protein interaction. The knowledge gained from this project would be invaluable for understanding the molecular mechanisms behind DNA replication initiation. Apart from its fundamental scientific importance, detailed insight into Mcm10 activities will be directly applicable to the development of novel anti-cancer agents targeting the DNA replication initiation machinery thus selectively eliminating cancer cells.

Publications

10 25 50
 
Title specific antibodies against human Mcm10 and various DNA constructs 
Description We have generated a specific polyclonal antibodies against human Mcm10. This is a significant tool that allows us now to investigate Mcm10 function in vivo and in vitro. We have generated a large panel of DNA constructs coding for Mcm10 domains and fragments designed for expression in various host systems. They all are tested now and produce stable soluble proteins. 
Type Of Material Antibody 
Year Produced 2009 
Provided To Others? Yes  
Impact We have shared our reagents with a number of collaboration partners in UK to accelerate our joint research. The Mcm10 antibody is unique as the commercial source is expensive and unreliable. Our expression constructs allow us and collaborators to speed up our study on structure-fuction relationship of Mcm10. 
 
Description Interaction between Mcm10 and SIRT1 histone deacetylase 
Organisation University of York
Department Department of Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution we identified SIRT1 as an interactive partner of Mcm10.
Collaborator Contribution siRNA design, antibodies and cell lines from the group with expertise in SIRT1
Impact this is strategic collaboration with the lab whose expertise is based on cell biology of SIRT1. having collaboration with this lab cut down time for experimental set up tremedously allowing us to focus on elucidating mechanism of SIRT1-dependent regulation of Mcm10
Start Year 2009
 
Description Mass spectrometry of Mcm10 acetylation sites 
Organisation University College London
Department Research Department of Primary Care & Population Health
Country United Kingdom 
Sector Academic/University 
PI Contribution we have supplied samples of acetylated (and deacetylated) Mcm10 and its domains
Collaborator Contribution Our collaborators provided mass spectroscopy analysis of our Mcm10 acetylated samples produced in vivo and vitro
Impact we have mapped most of lysine residues within Mcm10 sequence that are acetylated by p300 acetyltransferase and subsequently deacetylated by SIRT1
Start Year 2010
 
Description Mcm10 interaction with Cdc45 
Organisation Italian National Research Council
Department Institute of Biosciences and Bioresources
Country Italy 
Sector Public 
PI Contribution joint experiments on Mcm10-Cdc45 replication protein interaction and its importance in DNA replication initiation. Protein expression and purification, experiment planning, in vivo interaction, cell biology experiments, microscopy
Collaborator Contribution Cdc45-Mcm10 interaction, protein- DNA interaction
Impact "The physical interaction of Mcm10 with Cdc45 modulates their DNA binding properties" manuscript is under review in Nucleic Acid Research.
Start Year 2009
 
Description Protein-protein interaction between Mcm10 and Mcm2-7 
Organisation Birkbeck, University of London
Department Department of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution all collaborations were initiated by me to accelerate my research. The specific expertise and knowledge that these groups brought to my research is facilitating experimental work and analysis to be done in shortest time, allowing to avoid when possible a lengthy material and tools preparation step.
Collaborator Contribution studies on Mcm10 levels in cancer cellsStudies on Mcm10 and Mcm2-7 interaction and cooperative functionstructural studies of Mcm10. Electron microscopy of the full length Mcm10 protein and X-ray crystallography of its functional domains.Functional studies on Mcm10 interaction with DNA Polymerase alpha - primase complex.collaboration on using DNA fiber (combing) methodology to analyse Mcm10 role in DNA replication.Collaboration on Mcm10 interaction with chromatin remodelling factorsAn imporant DNA repair factor has been identified as Mcm10 interacting partner.
Impact these collaborations allowed (1) to complete quickly experiments on validation of Mcm10 as a target for drug-design by using DNA combing analyisis (with NCI, NIH). (2) two important Mcm10-interacting targets were identified through the collaboration with University of York and ICR, London. (3) Collaboration with Birkbeck college allows me to continue structural biology studies of Mcm10.
Start Year 2008
 
Description Protein-protein interaction between Mcm10 and Mcm2-7 
Organisation Institute of Cancer Research UK
Country United Kingdom 
Sector Academic/University 
PI Contribution all collaborations were initiated by me to accelerate my research. The specific expertise and knowledge that these groups brought to my research is facilitating experimental work and analysis to be done in shortest time, allowing to avoid when possible a lengthy material and tools preparation step.
Collaborator Contribution studies on Mcm10 levels in cancer cellsStudies on Mcm10 and Mcm2-7 interaction and cooperative functionstructural studies of Mcm10. Electron microscopy of the full length Mcm10 protein and X-ray crystallography of its functional domains.Functional studies on Mcm10 interaction with DNA Polymerase alpha - primase complex.collaboration on using DNA fiber (combing) methodology to analyse Mcm10 role in DNA replication.Collaboration on Mcm10 interaction with chromatin remodelling factorsAn imporant DNA repair factor has been identified as Mcm10 interacting partner.
Impact these collaborations allowed (1) to complete quickly experiments on validation of Mcm10 as a target for drug-design by using DNA combing analyisis (with NCI, NIH). (2) two important Mcm10-interacting targets were identified through the collaboration with University of York and ICR, London. (3) Collaboration with Birkbeck college allows me to continue structural biology studies of Mcm10.
Start Year 2008
 
Description Protein-protein interaction between Mcm10 and Mcm2-7 
Organisation National Cancer Institute (NCI)
Country United States 
Sector Public 
PI Contribution all collaborations were initiated by me to accelerate my research. The specific expertise and knowledge that these groups brought to my research is facilitating experimental work and analysis to be done in shortest time, allowing to avoid when possible a lengthy material and tools preparation step.
Collaborator Contribution studies on Mcm10 levels in cancer cellsStudies on Mcm10 and Mcm2-7 interaction and cooperative functionstructural studies of Mcm10. Electron microscopy of the full length Mcm10 protein and X-ray crystallography of its functional domains.Functional studies on Mcm10 interaction with DNA Polymerase alpha - primase complex.collaboration on using DNA fiber (combing) methodology to analyse Mcm10 role in DNA replication.Collaboration on Mcm10 interaction with chromatin remodelling factorsAn imporant DNA repair factor has been identified as Mcm10 interacting partner.
Impact these collaborations allowed (1) to complete quickly experiments on validation of Mcm10 as a target for drug-design by using DNA combing analyisis (with NCI, NIH). (2) two important Mcm10-interacting targets were identified through the collaboration with University of York and ICR, London. (3) Collaboration with Birkbeck college allows me to continue structural biology studies of Mcm10.
Start Year 2008
 
Description Protein-protein interaction between Mcm10 and Mcm2-7 
Organisation National Research Council
Department Institute of Protein Biochemistry
Country Italy 
Sector Public 
PI Contribution all collaborations were initiated by me to accelerate my research. The specific expertise and knowledge that these groups brought to my research is facilitating experimental work and analysis to be done in shortest time, allowing to avoid when possible a lengthy material and tools preparation step.
Collaborator Contribution studies on Mcm10 levels in cancer cellsStudies on Mcm10 and Mcm2-7 interaction and cooperative functionstructural studies of Mcm10. Electron microscopy of the full length Mcm10 protein and X-ray crystallography of its functional domains.Functional studies on Mcm10 interaction with DNA Polymerase alpha - primase complex.collaboration on using DNA fiber (combing) methodology to analyse Mcm10 role in DNA replication.Collaboration on Mcm10 interaction with chromatin remodelling factorsAn imporant DNA repair factor has been identified as Mcm10 interacting partner.
Impact these collaborations allowed (1) to complete quickly experiments on validation of Mcm10 as a target for drug-design by using DNA combing analyisis (with NCI, NIH). (2) two important Mcm10-interacting targets were identified through the collaboration with University of York and ICR, London. (3) Collaboration with Birkbeck college allows me to continue structural biology studies of Mcm10.
Start Year 2008
 
Description Protein-protein interaction between Mcm10 and Mcm2-7 
Organisation University College London
Department Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution all collaborations were initiated by me to accelerate my research. The specific expertise and knowledge that these groups brought to my research is facilitating experimental work and analysis to be done in shortest time, allowing to avoid when possible a lengthy material and tools preparation step.
Collaborator Contribution studies on Mcm10 levels in cancer cellsStudies on Mcm10 and Mcm2-7 interaction and cooperative functionstructural studies of Mcm10. Electron microscopy of the full length Mcm10 protein and X-ray crystallography of its functional domains.Functional studies on Mcm10 interaction with DNA Polymerase alpha - primase complex.collaboration on using DNA fiber (combing) methodology to analyse Mcm10 role in DNA replication.Collaboration on Mcm10 interaction with chromatin remodelling factorsAn imporant DNA repair factor has been identified as Mcm10 interacting partner.
Impact these collaborations allowed (1) to complete quickly experiments on validation of Mcm10 as a target for drug-design by using DNA combing analyisis (with NCI, NIH). (2) two important Mcm10-interacting targets were identified through the collaboration with University of York and ICR, London. (3) Collaboration with Birkbeck college allows me to continue structural biology studies of Mcm10.
Start Year 2008
 
Description Protein-protein interaction between Mcm10 and Mcm2-7 
Organisation University of York
Department Department of Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution all collaborations were initiated by me to accelerate my research. The specific expertise and knowledge that these groups brought to my research is facilitating experimental work and analysis to be done in shortest time, allowing to avoid when possible a lengthy material and tools preparation step.
Collaborator Contribution studies on Mcm10 levels in cancer cellsStudies on Mcm10 and Mcm2-7 interaction and cooperative functionstructural studies of Mcm10. Electron microscopy of the full length Mcm10 protein and X-ray crystallography of its functional domains.Functional studies on Mcm10 interaction with DNA Polymerase alpha - primase complex.collaboration on using DNA fiber (combing) methodology to analyse Mcm10 role in DNA replication.Collaboration on Mcm10 interaction with chromatin remodelling factorsAn imporant DNA repair factor has been identified as Mcm10 interacting partner.
Impact these collaborations allowed (1) to complete quickly experiments on validation of Mcm10 as a target for drug-design by using DNA combing analyisis (with NCI, NIH). (2) two important Mcm10-interacting targets were identified through the collaboration with University of York and ICR, London. (3) Collaboration with Birkbeck college allows me to continue structural biology studies of Mcm10.
Start Year 2008
 
Description Protein-protein interaction between Mcm10 and Mcm2-7 
Organisation University of York
Department Department of Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution all collaborations were initiated by me to accelerate my research. The specific expertise and knowledge that these groups brought to my research is facilitating experimental work and analysis to be done in shortest time, allowing to avoid when possible a lengthy material and tools preparation step.
Collaborator Contribution studies on Mcm10 levels in cancer cellsStudies on Mcm10 and Mcm2-7 interaction and cooperative functionstructural studies of Mcm10. Electron microscopy of the full length Mcm10 protein and X-ray crystallography of its functional domains.Functional studies on Mcm10 interaction with DNA Polymerase alpha - primase complex.collaboration on using DNA fiber (combing) methodology to analyse Mcm10 role in DNA replication.Collaboration on Mcm10 interaction with chromatin remodelling factorsAn imporant DNA repair factor has been identified as Mcm10 interacting partner.
Impact these collaborations allowed (1) to complete quickly experiments on validation of Mcm10 as a target for drug-design by using DNA combing analyisis (with NCI, NIH). (2) two important Mcm10-interacting targets were identified through the collaboration with University of York and ICR, London. (3) Collaboration with Birkbeck college allows me to continue structural biology studies of Mcm10.
Start Year 2008
 
Description Restructured the WIBR website 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Restructured the departamental website for the Wolfson Institute for Biomedical Research (WIBR) making it clear for general public. Currently re-organising the website for the Pathology department.

There an increased stream of contacts form general public, prospective students and researchers. The information on ongoing projects and research activities within the WIBR labarotories is clear and undesrstandable to general public and is constantly updated.
Year(s) Of Engagement Activity 2008,2009,2011,2013,2014
URL http://www.ucl.ac.uk/wibr
 
Description Wolfson Institute Brochure for general public 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Supervised the making of a brochure about the WIBR aimed to general public.

there an increased stream of contacts form general public, prospective students and researchers. The information on ongoing projects and research activities within the WIBR labarotories is clear and undesrstandable to general public.
Year(s) Of Engagement Activity 2008,2009