Structure of the Smc5-6 DNA repair and chromosome maintenance protein complex

Lead Research Organisation: University of Sussex
Department Name: Sch of Life Sciences

Abstract

The accurate inheritance of the genetic material (DNA) is key to the survival of all organisms. DNA has to be copied (replicated) before cells divide but obstacles such as damaged DNA bases can lead to problems during the copying process. Cells have developed different ways to remove such obstacles (repair), and if the DNA damage cannot be removed it can be bypassed, either during or immediately after DNA copying (tolerance). Failure of repair or tolerance can result in errors when the DNA is copied. These errors cause permanent genetic changes, which in turn can result in increased cancer incidence. Many proteins are involved in repair and tolerance. One of these, Smc5-6, which we have been studying for several years, is a big ?protein complex? with eight components. We propose to combine the expertise of our four research groups to determine the 3-dimensional structure of this complex. This will enable us to understand how the different components fit together, what they each do, and how they help to fulfil the functions of the protein complex in the cell. This will help us to understand how Smc6 acts to protect us from the problems caused by DNA damage during DNA copying. This work has implications for the development of cancer and aging in the general population.

Technical Summary

The SMC protein complexes, including cohesins and condensins, are required for maintenance of higher order chromosome structure and segregation. The Smc5/6 complex, the least well understood of the three SMC complexes, is required for DNA repair, rDNA stability and telomere maintenance. It consists of 8 subunits, which form three sub-complexes: the core Smc5 and Smc6 proteins with the SUMO ligase Nse2; Nse1-Nse3-Nse4, which includes the RING finger putative ubiquitin ligase Nse1; and Nse5-6. A further protein, Rad60 interacts transiently with the complex.

We wish to understand the in vivo functions of the Smc5/6 super-complex in terms of its biochemistry and its structure. In particular we wish to define the functional contributions of the individual subunits, determine how they assemble in the functional complex and sub-complexes, and elucidate the structural basis for regulation and coordination of the ATPase, SUMO ligase and ubiquitin E3 ligase activities, which the complex incorporates.

Towards this end we will develop expression and purification strategies for the assembled complex ex vivo from S.pombe, and the human and S.pombe complex, sub-complexes and individual proteins by recombinant expression in insect cells. We will use single-particle electron microscopy to define the overall architecture of the complex at low resolution, and combine this with high-resolution crystal structure determination of sub-complexes and individual proteins to achieve a quasi-atomic model for the full complex. In parallel, we will determine the ATPase, SUMO ligase and ubiquitin E3 ligase activities of the complex in the presence/absence of individual components (including Rad60), and with the inclusion of genetically characterised mutations in different components and domains for which the in vivo phenotype has already been defined. We will correlate these structural, biochemical and phenotypic data to define strong hypotheses for the roles of individual subunits and domains in the known functions of the Smc5/6 super-complex, and test these by epistasis and other genetic approaches in S.pombe. We will also use our structural and biochemical data in a reverse genetic approach to design targeted ?surgical? disruption of specific interfaces and interactions whose biochemical consequences can then be defined, with the aim of identifying separation-of-function mutations that affect only one of the biological processes to which Smc5/6 contributes.

These studies will provide profound insight into the structure, biochemistry and in vivo function of this important protein complex.

Publications

10 25 50
 
Description Development of treatment regimen for LICS syndrome
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
 
Description ISSF Live cell single-molecule imaging of DNA repair complexes in human cells
Amount £15,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2017 
End 09/2017
 
Description MRC project grant How do Smc5/6 interactions with DNA coordinate replication and recombination?
Amount £818,638 (GBP)
Funding ID MR/P018955/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 04/2017 
End 03/2020
 
Title fission yeast research methods 
Description research methods 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact dissemination of research methods to fission yeast community 
URL http://cshprotocols.cshlp.org/content/2016/5/
 
Description Collaboration with Central European Technology Institute 
Organisation Central European Institute of Technology (CEITEC)
Country Czech Republic 
Sector Charity/Non Profit 
PI Contribution Training of PhD student in techniques
Collaborator Contribution Conducting experiments associated with project
Impact Mapping of ubiquitylation sites on Nse4B
Start Year 2013
 
Description Collaboration with clinicians at University of Utrecht Medical Centre 
Organisation University Medical Center Utrecht (UMC)
Country Netherlands 
Sector Academic/University 
PI Contribution We have been able to provide structural and functional insight into the consequences of mutation in components of the NSE1/NSE3/NSE4 sub-complex of the Smc5/6 system, that occur in a newly described chromosome breakage syndrome associated with severe lung disease
Collaborator Contribution They identified and characterised the patients with this syndrome and determined the genetic mutations present
Impact A manuscript has now been accepted for publication in the Journal of Clinical Investigations.
Start Year 2014
 
Description circular chromosomes 
Organisation Hiroshima University
Country Japan 
Sector Academic/University 
PI Contribution Collaboration to determine genetic requirements for the maintenance of circular chromosomes. I provide the DNA damage response expertise and fission yeast genetics
Collaborator Contribution Collaborators provide telomere maintenance expertise and generate circular chromosomes in fission yeast
Impact publications PMID: 23297345, PMID: 29121084. Led to development of HiHA Hiroshima Healthy Aging research committee to promote research into healthy ageing at Hiroshima University. I am a member of the committee and attend an annual conference.
Start Year 2012
 
Description single molecule microscopy 
Organisation University of Sussex
Department School of Life Sciences Sussex
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of in vivo single molecule imaging of DNA repair proteins in human cells
Collaborator Contribution Development of PALM microscope and imaging analysis for in vivo single molecule imaging
Impact No outputs yet as ISSF funding awarded Feb 2017. Interdisciplinary collaboration between cell biologists and physical chemists
Start Year 2017
 
Title LICS syndrome treatment regimen 
Description We are at the very early stages of identifying individuals with LICS syndrome and ways of managing of the condition 
Type Management of Diseases and Conditions
Current Stage Of Development Initial development
Year Development Stage Completed 2017
Development Status Under active development/distribution
Impact Consultation between paediatricians and research scientists about possible treatment regimes and their likely impact given the genetic defect. This is ongoing and research results will both inform on treatment and lead to improved diagnostic tests. 
 
Description Brighton Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Hello Laurence.

Thank you for helping our DNA Day out with your recommendations. Oliver Wilkinson and Aaron Alt produced a terrific presentation which told the story of the history and discovery of the helical structure of DNA, Alison Pike's twin studies talk inspired a million questions, Alison Sinclair and her team gave a most entertaining display of synchronised gene expression and Adam Eyre-Walker was consummate in his analysis of evolution's future. We rounded off with Nessa Carey introducing us to epigenetics and the Professor Munir Primohamed of the British Pharmacological Society talking about personalised medicines. All of it was perfectly clear to a non-specialist audience, and they duly sat, perfectly still, through the whole five hours, and absorbed every word. I hope we will be able to come to you again in the future with similar challenges and come away with similar triumphs.

I'm sorry I haven't been in touch to thank you earlier. The Festival was a long one, and kept us very busy. I am only now emerging into the daylight, just in time for spring!

Best wishes

Richard



Richard Robinson
Brighton Science Festival
Richard@BrightonScience.com
01273 777 628
http://www.BrightonScience.com
18 Temple St, Brighton BN1 3BH
Want to know how the World will end? http://youtu.be/8YatZ0mrO40



General appreciation of importance of DNA
Year(s) Of Engagement Activity 2013
URL http://www.BrightonScience.com
 
Description Brighton Science Festival 2016 - Bacon and ?Eggs: The Journey to Cancer 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact University of Sussex, Genome Damage and Stability Centre: Bacon and Eggs: The Journey to Cancer All humans start off from a single fertilised cell, which duplicates itself to create the many trillions of cells contained within an adult human. But sometimes the duplication goes wrong - and this can lead to cancer. Is it all doom and gloom? Hopefully not in this light-hearted exploration of the science of cancer. The Genome Damage and Stability Centre is part of the School of Life Sciences, University of Sussex. GDSC - See more at: http://www.brightonscience.com/events/big-science-sunday/#sthash.iTfuP3oD.dpuf
Year(s) Of Engagement Activity 2016
 
Description press release on LICS syndrome 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Co-ordinated international (UK, Netherlands and USA) press release to publicise our study on LICS syndrome, an inherited syndrome associated with chromosome instability and immunodeficiency which leads to death in early childhood from lung disease. This has led to the identification of further affected families in the Netherlands and the US and the setting up of an advisory panel to guide treatment regimens
Year(s) Of Engagement Activity 2016