How do Smc5/6 interactions with DNA coordinate replication and recombination?

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

Abstract

AE1: DNA replication AE2: DNA repair double strand breaks/ Smc5/6 AE3: double-strand break repair mechanism AE4: SMC/cohesin-DNA protein structure

Technical Summary

Structural Maintenance of Chromosomes (SMC) complexes (condensin, cohesin and Smc5/6) play critical roles in orchestrating chromosome dynamics throughout the cell cycle and are essential for the maintenance of genomic integrity. Smc5/6 regulates homologous recombination and replication restart activities, both key to genome stability.
We have shown that the hinge-domain of Smc5/6 binds preferentially to single-strand DNA and that the NSMCE1/3/4 subcomplex binds preferentially to double-strand DNA (dsDNA). We propose that these two DNA-binding activities, separated by ~50nm, work together to control, regulate and ultimately resolve complex DNA structures such as homologous recombination and replication intermediates.
To investigate and test our hypothesis, we will co-crystallise the Smc5/6-hinge and NSMCE1/3/4 accessory complex with DNA, thus determining the molecular details underpinning each interaction type. For both complexes, we will also look at any concomitant changes in conformation driven by DNA-binding, using SAXS and other biophysical methods, such as FRET. With the structural information obtained, we will create and test defined mutations (in vitro and vivo) for effects on biological function.
We will isolate sufficient Smc5/6 holo-complex to enable single-particle electron microscopy and/or AFM-based approaches, examining the structure and conformational changes, in the presence and absence of bound DNA. We will determine if the holo-complex binds preferentially to biologically-relevant DNA structures, such as Holliday junctions, D-loops and fork-like structures, and whether this can occur in the presence of RPA (replication-protein A), and if DNA-interaction brings the head and hinge regions together.
These studies will provide insight into the structure, biochemistry and in vivo function of this important complex and lead to an understanding of how Smc5/6 coordinates replication and recombination and its role in maintaining human health.

Planned Impact

This project aims to elucidate how Smc5/6 interactions with DNA coordinate replication and recombination and to understand how such interactions affect Smc5/6 conformation and function. Thus, the project will benefit the Smc5/6 and SMC research communities. It will inform the wider DNA repair and recombination fields and has important implications for our understanding of related pathways in higher eukaryotes.

It will advance our understanding of the role Smc5/6 in maintaining human health. Smc5/6 DNA binding underpins its role in preventing lung disease, maintaining genetic stability and likely as a restriction factor for Hepatitis B infection. Therefore, the proposal will have significant impact for research progress in these areas, as well as contributing to the understanding of diseases related to DNA repair defects, such as cancer.

The potential to therapeutically modulate Smc5/6 function, for example to exploit synthetic interactions in cancer, will be enhanced by a detailed molecular understanding. It will also impact on treatments for Hepatitis B and potentially other viral infections. This work will thus contribute to improvements in the quality of life.

The proposal will also provide training for the next generation of scientists in cutting edge techniques and transferable skills, impacting on the creation of a skilled workforce.

Lastly, by developing collaborations between researchers in different disciplines and Schools it will impact on the development of inter-disciplinary research.

Publications

10 25 50
publication icon
Baxter J (2019) Are SMC Complexes Loop Extruding Factors? Linking Theory With Fact. in BioEssays : news and reviews in molecular, cellular and developmental biology

publication icon
Murray J (2018) Integrating DNA damage repair with the cell cycle in Current Opinion in Cell Biology

 
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 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
 
Description SMC Proteins - Japan - 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Dr Johanne Murray delivered a short talk describing the experimental work, and working models, that have been generated at the GDSC, Sussex - regarding the Smc5/6 complex
Year(s) Of Engagement Activity 2017
URL http://www.smcproteins2017.org