Smc5/6 and replication fork stability

Lead Research Organisation: University of Sussex
Department Name: Brighton and Sussex Medical School

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 and DNA-proteins complexes can lead to replication stalling and breaking down. Cells have developed a range of mechanisms to remove such obstacles and if the DNA damage cannot be removed it can be bypassed either during or immediately after DNA copying by a range of mechanisms. This is important for survival but can be at the expense of an increased error rate leading to mutations and an increase in genome rearrangements. Since such changes can lead to cancer it is vital that copying is coordinated with repair and restarts correctly after stalling.

The Smc6 protein is essential for cell survival in all organisms from yeast to humans. Our work in yeast suggests that Smc6 has an important role in coordinating the repair of DNA damage with the copying of the DNA which must be accurate so that each new cell contains identical genetic material. When Smc6 is not fully functional cells use inappropriate processes to repair or tolerate the DNA damage and this leads to an increase in copying errors, which can result in changes to the genome and sometimes to cell death.

We want to understand how Smc6 acts to protect us from the deleterious effects of DNA damage during DNA copying because this has implications for the development of cancer and aging in the general population. Because yeast cells repair and copy their DNA in a similar way to human cells, and because yeast is easy and cheap to manipulate, we can use yeast to study how the Smc6 protein associates with the DNA copying apparatus and the DNA itself, and establish if and how it coordinates the activity of other factors to ensure copying restarts correctly.

Technical Summary

Replication fork stability is vital for cell and organismal survival. Homologous recombination (HR) rescues collapsed forks, but at the potential expense of genome instability. In humans HR proteins are essential for S phase progression and defects in regulating HR, i.e. in Bloom?s syndrome, lead to increased cancer susceptibility. This underscores the importance of HR pathways for human health. We recently showed that the essential (and highly conserved) Smc5/6 complex is required to recruit HR proteins to stable stalled replication forks.
SMC complexes, including Cohesins and Condensins, are required for higher order chromosome structure and segregation. The Smc5/6 complex is the least well understood. Phenomenologically, Smc5/6 is required for HR, rDNA stability and telomere maintenance. By characterising Schizosaccharomyces pombe smc6 mutants, we defined two separate Smc5/6 functions in HR. First, most smc6 mutants are defective in processing recombination-dependent DNA intermediates when replication forks collapse. This leads to defects in chromosome segregation and increased rDNA recombination: a ?late? role in HR.
Second, we showed that Smc5/6 is required to load Rpa and Rad52 when replication stalls and so keep such stalled forks primed for resumption. This new ?early? role for Smc5/6 is defective specifically in the smc6-74 mutant, which maps to a conserved ?arginine finger?. In bacterial Smc proteins this domain is required for DNA-dependent ATP hydrolysis, likely to regulate opening and closing of Smc head domains. Defects in the early role correlated with increased rDNA stability. Thus Smc5/6 regulation of HR is important for replication resumption when forks are stalled but comes at the expense of increased genome instability. This function is particularly important in regions of unidirectional replication.
This proposal seeks to define the mechanism by which Smc5/6 regulates HR at stalled forks. We will determine:
? If DNA-dependent ATP hydrolysis is required for fork stability. Smc5/6 complexes will be purified and ATP hydrolysis analysed in relevant mutants.
? If other Smc5/6 domains/enzymatic activities are required. Smc6 mutants that suppress rDNA recombination will be identified, their responses to DNA damage and their ATPase activity analysed.
? Whether Smc5/6 functions directly or indirectly. Smc5/6 has been implicated in loading cohesins after DNA damage. The chromatin loading of the other Smc complexes upon replication stalling will be characterised in relevant smc6 mutants.
? If previously active replication forks resume replication efficiently (genome-wide and at the rDNA) or if new replication origins fire to complete S phase in smc6 mutants.
 
Description Development of treatment regimen for LICS syndrome
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
 
Description EMBO course 2012
Geographic Reach Asia 
Policy Influence Type Influenced training of practitioners or researchers
Impact Training of research scientists increases their effectiveness in discovery science. Such research underpins translational research and thus in the long term contributes to increasing the quality of life.
 
Description BBSRC project grant Repair of replication-associated double strand breaks
Amount £500,000 (GBP)
Funding ID BB/K019805/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2016
 
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
 
Description MRC project grant Structure of the Smc5-6 DNA repair and chromosome maintenance protein complex
Amount £686,445 (GBP)
Funding ID G1001668 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2012 
End 12/2014
 
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/
 
Title methods for working with the model organism S. pombe 
Description Contributed to a laboratory manual for working with fission yeast 
Type Of Material Improvements to research infrastructure 
Year Produced 2016 
Provided To Others? Yes  
Impact publication of methods manual, training of a skilled research workforce 
 
Title Omero 
Description Storage and analysis of images from fixed cell or live cell imaging 
Type Of Material Data handling & control 
Provided To Others? No  
Impact Secure backed up storage and analysis of microscopy images. 
 
Title R drive 
Description storage of research data on secure backed up server 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact secure storage of research data 
 
Description Smc5/6 
Organisation Icahn School of Medicine at Mount Sinai
Department Department of Oncological Sciences
Country United States 
Sector Academic/University 
PI Contribution reagents, methodologies and intellectual contribution
Collaborator Contribution access to reagents
Impact joint publications
Start Year 2006
 
Description Smc5/6 clinical genetics 
Organisation Baylor College of Medicine
Country United States 
Sector Hospitals 
PI Contribution We have characterised the protein levels, molecular and cellular defects in fibroblasts derived from patients with mutations in NSMCE3 encoding one of the subunits of the Smc5/6 complex
Collaborator Contribution Clinicians and clinical geneticists at Baylor and UMC identified patients with a novel chromosome breakage syndrome which by whole genome sequencing was found to be due to mutation in NSMCE3. Our colleagues in Masaryk university, Brno, carried out the yeast two hybrid analysis to support or analysis.
Impact This is a multidisciplinary collaboration between clinicians, clinical geneticists and discovery scientists. The clinical groups in the Netherlands and the US were brought together through GENEMATCHER which facilitates collaboration between groups working on syndromes due to mutations in the same gene. Both groups identified mutations in the same gene which led to very similar phenotypes and combining the data has doubled the number of affected individuals studied. It is a good example of how powerful GENEMATCHER is as resource. We were brought into the collaboration as experts in the Smc5/6 complex in order to identify the defects at the cellular level. We have been able to correlate the defect in the patient cells with destabilisation of the Smc5/6 complex and a defect in homologous recombination. It benefits our research as we now have defined cell lines in which to study the roles of the Smc5/6 complex. It also benefits the health care specialists as we have also developed high throughput assays to study the cellular defects and these can be used as diagnostic assays.
Start Year 2014
 
Description Smc5/6 clinical genetics 
Organisation Masaryk University
Country Czech Republic 
Sector Academic/University 
PI Contribution We have characterised the protein levels, molecular and cellular defects in fibroblasts derived from patients with mutations in NSMCE3 encoding one of the subunits of the Smc5/6 complex
Collaborator Contribution Clinicians and clinical geneticists at Baylor and UMC identified patients with a novel chromosome breakage syndrome which by whole genome sequencing was found to be due to mutation in NSMCE3. Our colleagues in Masaryk university, Brno, carried out the yeast two hybrid analysis to support or analysis.
Impact This is a multidisciplinary collaboration between clinicians, clinical geneticists and discovery scientists. The clinical groups in the Netherlands and the US were brought together through GENEMATCHER which facilitates collaboration between groups working on syndromes due to mutations in the same gene. Both groups identified mutations in the same gene which led to very similar phenotypes and combining the data has doubled the number of affected individuals studied. It is a good example of how powerful GENEMATCHER is as resource. We were brought into the collaboration as experts in the Smc5/6 complex in order to identify the defects at the cellular level. We have been able to correlate the defect in the patient cells with destabilisation of the Smc5/6 complex and a defect in homologous recombination. It benefits our research as we now have defined cell lines in which to study the roles of the Smc5/6 complex. It also benefits the health care specialists as we have also developed high throughput assays to study the cellular defects and these can be used as diagnostic assays.
Start Year 2014
 
Description Smc5/6 clinical genetics 
Organisation University Medical Center Utrecht (UMC)
Country Netherlands 
Sector Academic/University 
PI Contribution We have characterised the protein levels, molecular and cellular defects in fibroblasts derived from patients with mutations in NSMCE3 encoding one of the subunits of the Smc5/6 complex
Collaborator Contribution Clinicians and clinical geneticists at Baylor and UMC identified patients with a novel chromosome breakage syndrome which by whole genome sequencing was found to be due to mutation in NSMCE3. Our colleagues in Masaryk university, Brno, carried out the yeast two hybrid analysis to support or analysis.
Impact This is a multidisciplinary collaboration between clinicians, clinical geneticists and discovery scientists. The clinical groups in the Netherlands and the US were brought together through GENEMATCHER which facilitates collaboration between groups working on syndromes due to mutations in the same gene. Both groups identified mutations in the same gene which led to very similar phenotypes and combining the data has doubled the number of affected individuals studied. It is a good example of how powerful GENEMATCHER is as resource. We were brought into the collaboration as experts in the Smc5/6 complex in order to identify the defects at the cellular level. We have been able to correlate the defect in the patient cells with destabilisation of the Smc5/6 complex and a defect in homologous recombination. It benefits our research as we now have defined cell lines in which to study the roles of the Smc5/6 complex. It also benefits the health care specialists as we have also developed high throughput assays to study the cellular defects and these can be used as diagnostic assays.
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? No
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Participate in running a stall at the Brighton Science Festival with a range of primary school childrens activities and more advanced displays aimed at explaining an aspect of biology to children and parents

Generated interesting discussions
Year(s) Of Engagement Activity 2009,2010,2011,2012
 
Description MRC centenary open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Science/art fusion Open day consisting of talks and hands on exhibitions showcasing a bench to bedside approach to genome stability with a parallel exhibition of art installations by internationally recognized artists, was attended by secondary school teachers, health care professionals, patient groups and the lay public.

Interest expressed by schools in using videos and posters for GCSE and A level students. Discussion of putting together a roadshow of elements of open day.
Year(s) Of Engagement Activity 2013
URL http://mrc-genome-openday.com/#!/
 
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
 
Description school work experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Schools
Results and Impact four year 10 students from locals schools attended a week of intensive training in cancer research. They were given background talks and learned a range of laboratory techniques including genetics, cell biology, live cell imaging, western blotting and restriction fragment analysis. Their results were then discussed in relation to cancer research.

Very positive response from students who have kept in touch and continue to ask advise on careers in science.
Year(s) Of Engagement Activity 2009,2010,2011,2012