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.
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.
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.
Organisations
- University of Sussex (Lead Research Organisation)
- Biotechnology and Biological Sciences Research Council (Co-funder)
- Masaryk University (Collaboration)
- University of Sussex (Collaboration)
- Baylor College of Medicine (Collaboration)
- Icahn School of Medicine at Mount Sinai (Collaboration)
- University Medical Center Utrecht (UMC) (Collaboration)
- UNIVERSITY OF KENT (Collaboration)
Publications
Murray JM
(2018)
Integrating DNA damage repair with the cell cycle.
in Current opinion in cell biology
Baxter J
(2019)
Are SMC Complexes Loop Extruding Factors? Linking Theory With Fact.
in BioEssays : news and reviews in molecular, cellular and developmental biology
Vondrova L
(2020)
A role of the Nse4 kleisin and Nse1/Nse3 KITE subunits in the ATPase cycle of SMC5/6.
in Scientific reports
Liu Y
(2021)
The intra-S phase checkpoint directly regulates replication elongation to preserve the integrity of stalled replisomes.
in Proceedings of the National Academy of Sciences of the United States of America
Etheridge TJ
(2021)
Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo.
in eLife
Hallett ST
(2021)
Nse5/6 is a negative regulator of the ATPase activity of the Smc5/6 complex.
in Nucleic acids research
Grange LJ
(2022)
Pathogenic variants in SLF2 and SMC5 cause segmented chromosomes and mosaic variegated hyperploidy.
in Nature communications
Hallett ST
(2022)
Cryo-EM structure of the Smc5/6 holo-complex.
in Nucleic acids research
Description | Single molecule studies of reconstituted Smc5/6 complexes |
Organisation | University of Kent |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Provision of purified, reconstituted Smc5/6 complexes - with defined subunit compositions |
Collaborator Contribution | Single-molecule experiments on DNA-tightropes |
Impact | None |
Start Year | 2019 |
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 | Genome organisation by SMC complex (Biochemical Society) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Selected speaker (from abstract) at international conference. |
Year(s) Of Engagement Activity | 2022 |
Description | Institut Curie - Paris |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Other audiences |
Results and Impact | Invited speaker, Seminar Programme |
Year(s) Of Engagement Activity | 2022 |
Description | Invited Speaker - University of Glasgow |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | Scientific talk as part of an organised seminar programme |
Year(s) Of Engagement Activity | 2023 |
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 |
Description | Scientific Talk, Invited Speaker - University of Kent |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | Scientific talk as part of an organised seminar programme |
Year(s) Of Engagement Activity | 2023 |
Description | Talk / Presentation |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | Invited Speaker / Seminar Program / Leeds University / Astbury Centre |
Year(s) Of Engagement Activity | 2022 |