DNA Replication

Lead Research Organisation: MRC Clinical Sciences Centre

Abstract

The duplication of the genome has to function in a precise regulated way with errors resulting potentially in promoting cancer and other diseases. A multi-protein machine, which performs the duplication process, assembles onto DNA every time the DNA is duplicated and consequently disassembles once the process is finished. We study the process of the assembly pathway, which is frequently misregulated in cancer. To analyze the DNA duplication mechanisms we are using yeast as a model system, since the major DNA duplication mechanism are conserved from yeast to human and yeast has genetically defined start sites on the DNA to initiate DNA duplication. We use biochemical and structural methods to understand the properties and shape of the proteins and the DNA involved. We are specifically interested in how the protein machine chooses the perfect spot on the DNA for assembly, how certain proteins function to load a ring shaped protein complex onto the rod shaped DNA and the consequences to the protein-DNA interface. Consequently we want to understand the function of the round shape protein complex on the DNA, which potentially promotes separation of the 2 strands of DNA during duplication. Also we would like to understand more about the regulation of the DNA duplication process, where some regulators are known to be misregulated in cancer. Therefore we are interested how regulators influence key processes during assembly, which we monitor to address their role.

Technical Summary

The precise duplication of chromosomal DNA is essential to preserve the genetic complement of the cell. In multicellular organisms mistakes during genetic inheritance can lead to a cell population that proliferates uncontrolled. To ensure that chromosomes only replicate once per cell cycle the process of chromosome duplication is divided into discrete steps which have to happen in a specific order for successful assembly of a DNA replication machine. The first major step is licensing of chromosomes during late in mitosis or early G1 phase. This reaction involves i.) Binding of the six-subunit Origin Recognition Complex (ORC) to origin DNA and ii.) Cdc6 and Cdt1 dependent loading of Mini-Chromosome maintenance proteins (MCM), a putative DNA helicase, onto chromatin to form the pre-replicative complex (pre-RC). Once the cell is committed to cell division and DNA replication, Dbf4 Dependent Kinase (DDK) and Cyclin Dependent Kinases (CDKs) get activated. In the second major step several proteins are recruited to origins, including Replication Protein A (RPA) and DNA polymerases, which lead to pre-initiation complex (pre-IC) formation and DNA replication. Origins that replicated get inactivated by S-phase specific cyclins to restrict DNA replication to once per cell cycle.||We are interested in understanding the function, mechanism and regulation of the multiprotein machine that assembles in a multistep process on DNA resulting in duplication of the genome. To address these questions we use yeast as a model system, since it is the only biochemical accessible system available that allow sequence specific complex assembly and will serve as good basis for a general understanding of eukaryotic DNA replication. The 1st aim is to determine the basic mechanisms in assembly and regulation of the preRC. Recently we analyzed the role of ORC and Cdc6 in pre-RC formation. Now our focus will be on Cdt1 and MCM proteins in pre-RC formation. We will analyze the interactions between Cdt1-MCM2-7 and ORC/Cdc6 in solution and on DNA using biochemical assays. We are interested in the question how Cdt1-MCM2-7 modifies the previously studied ORC-Cdc6-DNA complex. To analyze the function of MCM proteins we do assemble the entire pre-RC and also understand the binding/loading of MCM proteins. In collaboration with Huilin Li (Brokkhaven National Laboratory, USA) we will determine the 3D structures of multiple complexes on DNA with ORC, Cdc6, Cdt1 and MCM proteins. Structural and biochemical data will be consequently integrated into a model describing the function and mechanism in pre-RC formation. This information will be used to understand the consequences of pre-RC misregulation in cancer.

Publications


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Chen Z (2008) The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae. in Proceedings of the National Academy of Sciences of the United States of America
Evrin C (2009) A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication. in Proceedings of the National Academy of Sciences of the United States of America
 
Description DFG Postdoctoral Fellowship
Amount £25,000 (GBP)
Organisation German Research Foundation (DFG) 
Sector Public
Country Germany, Federal Republic of
Start 10/2012 
End 09/2013
 
Description DFG Postdoctoral Fellowship
Amount £25,000 (GBP)
Organisation German Research Foundation (DFG) 
Sector Public
Country Germany, Federal Republic of
Start 03/2010 
End 04/2011
 
Description MRC Millenium Award
Amount £40,000 (GBP)
Organisation MRC-UK 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 08/2012 
End 07/2013
 
Title Antibodies against replication factors 
Description We have developed several polyclonal peptide antibodies against replication factors. 
Type Of Material Antibody 
Provided To Others? No  
Impact Now we can detect small amounts of proteins in complex reaction mixtures, which is vital to discover the functions and mechanism of the proteins. 
 
Title Purification of MCM2-7 helicase 
Description To study the role of the ORC-Cdc6 complex in DNA helicase loading we have developed a unique expression and purification scheme for the helicase. The DNA helicase, a central protein in initiation of DNA replication, acts at the DNA replication fork and consists of 6 subunits MCM2-7. Purification schemes have been published, but resulted in non-homogenous preparations containing MCM2-7 and MCM4,6,7. In addition these preparations contained mixtures of single hexameric complexes and double-hexameres. Our purification is containing only the single hexamer and consists of MCM2-7 without any MCM4,6,7. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact This preparation will allow functional characterisation of MCM2-7 and structural analysis in collaboration with a structural EM group. 
URL http://www.ncbi.nlm.nih.gov/pubmed/19910535
 
Title Reconstitution of pre-RC formation with MCM2-7 
Description We have reconstituted the loading of the MCM2-7 helicase on DNA in vitro using origin DNA, purified ORC, Cdc6, Cdt1 and MCM2-7 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2009 
Provided To Others? Yes  
Impact This mechanism forms the basis of future research and our conducted assays showed that the MCM2-7 helicase assembled into a double-hexamer and can slide on DNA. 
URL http://www.ncbi.nlm.nih.gov/pubmed/19910535
 
Title Sld3 
Description We have developed a purification strategy for the yeast replication factor Sld3 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Now that we can purify the factor we can test the activity in our assays. 
 
Description Bruce Stillman 
Organisation Cold Spring Harbor Laboratory (CSHL)
Country United States of America 
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 Fadri 
Organisation Medical Research Council (MRC)
Department MRC Clinical Sciences Centre
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution We have performed biochemical analysis
Collaborator Contribution The collaborator, Dr. Enrique Martinez-Perez, has performed in vivo analysis.
Impact Silva N, Ferrandiz N, Barroso C, Tognetti S, Lightfoot J, Telecan O, Encheva V, Faull P, Hanni S, Furger A, Snidjers B, Speck C and Martinez-Perez E (2014) The fidelity of synaptonemal complex assembly is regulated by a signaling mechanism that controls early meiotic progression Developmental Cell, Nov 24;31(4):503-11 - IF 10.4
Start Year 2013
 
Description Huilin Li 
Organisation Van Andel Institute
Country United States of America 
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 of Great Britain & Northern Ireland (UK) 
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 Michael 
Organisation Van andel Research Institute (VARI)
Country United States of America 
Sector Academic/University 
PI Contribution We have performed biochemical assays with Cdc6 and its mutants.
Collaborator Contribution Our collaborator has performed in vivo assays with Cdc6 and its mutants.
Impact 26305410 A co-authored publication: Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication FuJung Chang, Alberto Riera, Cecile Evrin, Jingchuan Sun, Huilin Li, Christian Speck, Michael Weinreich
Start Year 2010
 
Description Rudi Lurz 
Organisation Max Planck Society
Department Max Planck Institute for Molecular Genetics
Country Germany, Federal Republic of 
Sector Public 
PI Contribution We prepared samples for analysis.
Collaborator Contribution Electron microscopy preparation and analysis of samples
Impact 19910535, 24234446, 23376927
Start Year 2008
 
Description BPoD 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact To inform, engage the public and to raise the profile of the MRC/university
Year(s) Of Engagement Activity 2015
URL http://bpod.mrc.ac.uk/archive/2015/8/28
 
Description DNA Replication - How everything starts! 
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 Hosted a visit of the university of 3rd age

The audiance engaged with the science and appreciated the presentation.
Year(s) Of Engagement Activity 2007
 
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