Understanding how ribonucleotide reductase is regulated by the intrinsically disordered protein Spd1
Lead Research Organisation:
University of Oxford
Department Name: Zoology
Abstract
A basic step required for cell division is the replication of the genetic material, DNA. Replication of DNA requires dNTP building blocks and these not are required for chromosome duplication, but are also involved in other chromosomal processes such as DNA repair and recombination. An important step in the production of dNTPs involves an enzyme called ribonucleotide reductase, which catalyzes the rate limiting step in the pathway. Ribonucleotide reductase must be switched on to allow chromosome replication, as the pool of free dNTPs in cells is only sufficient for replicating a small fraction of the genome. It is also important that ribonucleotide reductase is not switched on all the time, as high concentrations of dNTPs reduces the fidelity of DNA polymerases which copy DNA, generating mutations which are harmful to the cell. As ribonucleotide reductase has such a pivotal role, drugs which inhibit this enzyme are useful in medicine as, by inhibiting DNA replication, they can slow down cell proliferation, and this can be useful in the treatment of cancer and other diseases where slowing cell division is beneficial.
Ribonucleotide reductase is regulated in the cell in several ways, reflecting the importance of this process. One mechanism involves small protein inhibitors which bind to ribonucleotide reductase. These protein inhibitors have so far only been found in yeasts, but there are good reasons to expect that they function also in human cells.
In this grant we will study a specific inhibitor of ribonucleotide reductase called Spd1 which is found in fission yeast. Although Spd1 is known to inhibit ribonucleotide reductase, the way that it does this is not well understood. We will investigate the mechanism of Spd1 inhibition using methods that can be used to detect interaction between Spd1 and the subunits of ribonucleotide reductase in living cells. Using yeast genetics we go onto select for ribonucleotide reductase mutants that are resistant to Spd1 inhibition, and analysis of these mutants should shed light on the mechanism of Spd1 inhibition. Specifically we can look at where these mutations occur in the structure of RNR, and this may help to define a binding surface for Spd1 on the enzyme. Ribonucleotide reductase is highly conserved in evolution, and we will explore whether Spd1 can inhibit ribonucleotide reductase from cells from multicellular organisms, which will be a step to determining whether similar protein inhibitors function in human cells. Understanding this inhibitory mechanism could be practically useful, as it may suggest novel ways of developing ways of inhibiting ribonucleotide reductase, thus extending the range of currently available drugs.
Ribonucleotide reductase is regulated in the cell in several ways, reflecting the importance of this process. One mechanism involves small protein inhibitors which bind to ribonucleotide reductase. These protein inhibitors have so far only been found in yeasts, but there are good reasons to expect that they function also in human cells.
In this grant we will study a specific inhibitor of ribonucleotide reductase called Spd1 which is found in fission yeast. Although Spd1 is known to inhibit ribonucleotide reductase, the way that it does this is not well understood. We will investigate the mechanism of Spd1 inhibition using methods that can be used to detect interaction between Spd1 and the subunits of ribonucleotide reductase in living cells. Using yeast genetics we go onto select for ribonucleotide reductase mutants that are resistant to Spd1 inhibition, and analysis of these mutants should shed light on the mechanism of Spd1 inhibition. Specifically we can look at where these mutations occur in the structure of RNR, and this may help to define a binding surface for Spd1 on the enzyme. Ribonucleotide reductase is highly conserved in evolution, and we will explore whether Spd1 can inhibit ribonucleotide reductase from cells from multicellular organisms, which will be a step to determining whether similar protein inhibitors function in human cells. Understanding this inhibitory mechanism could be practically useful, as it may suggest novel ways of developing ways of inhibiting ribonucleotide reductase, thus extending the range of currently available drugs.
Technical Summary
Ribonucleotide reductase (RNR) is an enzyme that catalyzes the rate-limiting step in the production of building blocks (dNTPs) for DNA replication. RNR is composed of complexes of two subunits, the large R1 catalytic subunit, and the smaller R2 subunit that provides the radical necessary for nucleotide reduction. RNR is subject to exquisite regulation to ensure that the concentration of dNTPs is optimal for high fidelity DNA synthesis. In this proposal, we will clarify the mode of action of a specific RNR inhibitor, Spd1 (for S phase delayed, referring to the effect of over-expressing Spd1) of Schizosaccharomyces pombe. Our experiments will address the following points:
- Determine which subunit of RNR Spd1 interacts with in vivo using bimolecular fluorescence complementation and antibody proximity (Duolink) methodologies.
- Determine if Spd1 affects interaction between R1 and R2 subunits, to try to determine if the oligomerization status of the enzyme is affected.
- Using strains where Spd1, R1 and R2 subunits are engineered to be predominantly nuclear, determine if Spd1 can affect RNR activity to determine if Spd1's effects on cellular compartmentalisation of RNR subunits is important for its mechanism.
- Obtain RNR mutants where Spd1 inhibition is abolished, thus identifying key residues, and a binding surface in the 3D RNR structure, involved in RNR regulation
- Establish whether ability of Spd1 to interact with the replication/repair protein PCNA as well as RNR means that it has the capacity to direct RNR to sites of DNA synthesis, which would make sense in terms of synthesizing dNTPS at sites where they are needed.
- Follow up preliminary observations that large complexes of Spd1 and R1 can be detected in live cells. Are they relevant to the regulation of RNR and does their appearance correlate with RNR activity or inactivity?
- Determine if Spd1 can inhibit metazoan RNR.
- Determine which subunit of RNR Spd1 interacts with in vivo using bimolecular fluorescence complementation and antibody proximity (Duolink) methodologies.
- Determine if Spd1 affects interaction between R1 and R2 subunits, to try to determine if the oligomerization status of the enzyme is affected.
- Using strains where Spd1, R1 and R2 subunits are engineered to be predominantly nuclear, determine if Spd1 can affect RNR activity to determine if Spd1's effects on cellular compartmentalisation of RNR subunits is important for its mechanism.
- Obtain RNR mutants where Spd1 inhibition is abolished, thus identifying key residues, and a binding surface in the 3D RNR structure, involved in RNR regulation
- Establish whether ability of Spd1 to interact with the replication/repair protein PCNA as well as RNR means that it has the capacity to direct RNR to sites of DNA synthesis, which would make sense in terms of synthesizing dNTPS at sites where they are needed.
- Follow up preliminary observations that large complexes of Spd1 and R1 can be detected in live cells. Are they relevant to the regulation of RNR and does their appearance correlate with RNR activity or inactivity?
- Determine if Spd1 can inhibit metazoan RNR.
Planned Impact
Academic impact:
Enhancing the knowledge economy - This research addresses regulation of an enzyme that is key for DNA synthesis and repair and thus should generate new information relating to how genome stability is maintained during many cycle of chromosome replication.
Beneficiaries will be students and biomedical workers.
Delivering and training highly skilled researchers - The research programme will contribute to developing the skill and training of the employed RA and student workers in the group.
Economic and societal impact:
Exploitation of scientific knowledge - The focus of this grant is on an ribonucleotide reductase, an enzyme which is a drug target for treatment of cancer and myeloproliferative diseases. It is possible that in the long term, results from this grant will contribute to the development of new drugs to complement existing inhibitors of ribonucleotide reductase. Also this work may shed light on how defects in ribonucleotide reductase regulation can lead to genome instability, which potentially can lead to cancer and ageing defects. Defects in the p53R2 subunit of ribonucleotide reductase cause diseases due mitochondrial DNA depletion syndrome and it is likely that other ribonucleotide reductase regulatory defects will be discovered.
Beneficiaries will potentially be members of the general public who would benefit from development of new drugs and medical advances relating to understanding ribonucleotide reductase regulation.
The work is relevant to these specific BBSRC strategic priorities:
- Basic bioscience underpinning health: ageing research, lifelong health and wellbeing - Insight into RNR regulation is relevant to how genome stability is maintained through development and in adult life
- Technology development for the biosciences - Bimolecular fluorescence complementation technology has been little used in the fission yeast model organism and we hope that our work will help demonstrate the usefulness of this method
Enhancing the knowledge economy - This research addresses regulation of an enzyme that is key for DNA synthesis and repair and thus should generate new information relating to how genome stability is maintained during many cycle of chromosome replication.
Beneficiaries will be students and biomedical workers.
Delivering and training highly skilled researchers - The research programme will contribute to developing the skill and training of the employed RA and student workers in the group.
Economic and societal impact:
Exploitation of scientific knowledge - The focus of this grant is on an ribonucleotide reductase, an enzyme which is a drug target for treatment of cancer and myeloproliferative diseases. It is possible that in the long term, results from this grant will contribute to the development of new drugs to complement existing inhibitors of ribonucleotide reductase. Also this work may shed light on how defects in ribonucleotide reductase regulation can lead to genome instability, which potentially can lead to cancer and ageing defects. Defects in the p53R2 subunit of ribonucleotide reductase cause diseases due mitochondrial DNA depletion syndrome and it is likely that other ribonucleotide reductase regulatory defects will be discovered.
Beneficiaries will potentially be members of the general public who would benefit from development of new drugs and medical advances relating to understanding ribonucleotide reductase regulation.
The work is relevant to these specific BBSRC strategic priorities:
- Basic bioscience underpinning health: ageing research, lifelong health and wellbeing - Insight into RNR regulation is relevant to how genome stability is maintained through development and in adult life
- Technology development for the biosciences - Bimolecular fluorescence complementation technology has been little used in the fission yeast model organism and we hope that our work will help demonstrate the usefulness of this method
People |
ORCID iD |
Stephen Kearsey (Principal Investigator) |
Publications
Pai CC
(2017)
A Critical Balance: dNTPs and the Maintenance of Genome Stability.
in Genes
Rayner E
(2016)
A panoply of errors: polymerase proofreading domain mutations in cancer.
in Nature reviews. Cancer
Pai CC
(2019)
An essential role for dNTP homeostasis following CDK-induced replication stress.
in Journal of cell science
Guarino E
(2014)
Cellular regulation of ribonucleotide reductase in eukaryotes.
in Seminars in cell & developmental biology
Aoude LG
(2015)
POLE mutations in families predisposed to cutaneous melanoma.
in Familial cancer
Guarino E
(2014)
Real-time imaging of DNA damage in yeast cells using ultra-short near-infrared pulsed laser irradiation.
in PloS one
Description | dNTP levels measurements in S. pombe cells by HPLC |
Organisation | University of Oxford |
Department | CRUK/MRC Oxford Institute for Radiation Oncology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | I provided cell samples that were used to extract dNTPs and to measure their levels by using HPLC technology. A specific protocol was developed and standardized, which is now being used routinely. |
Collaborator Contribution | The data on dNTP levels in different strains and conditions were vital for our research, and this collaboration provided this important information. |
Impact | This collaboration provided us technical assistance in the measurement of cellular dNTP levels. Development of a working protocol for dNTP measurement in S. pombe cells, that is fast and does not require the use of radioactivity. |
Start Year | 2015 |
Description | 6th Annual ShanghaiTech Biofrontier (Genome Engineering), Shanghai, China (19-20 October 2018) (Talk and Poster) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Professional Practitioners |
Results and Impact | 6th Annual ShanghaiTech Biofrontier (Genome Engineering), Shanghai, China (19-20 October 2018) (Talk and Poster) |
Year(s) Of Engagement Activity | 2018 |
Description | Oral and Poster presentation at "The 4th Annual ShanghaiTech BioFrontier: Frontier of Cell Biology" (16-17 December 2016, Shanghai, China) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | The conference held in Shanghai, is organised by ShanghaiTech University every year and the delegates were from all over China and Hong Kong. Dr Andreadis presented the BBSRC supported work initially as a poster - this was awaded best poster prize and Dr Andreadis was selected to present the work as a talk. |
Year(s) Of Engagement Activity | 2013,2016 |
Description | Poster presentation at 8th International Fission Yeast meeting, Kobe, Japan |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Poster presentation on current research. |
Year(s) Of Engagement Activity | 2015 |
Description | Poster presentation at Oxford Interisciplinary Bioscience Networking Event, Oxford, March 2015 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Industry/Business |
Results and Impact | Graduate students and early career researchers who were funded by the BBSRC participated in this event. It's aim was to bring in contact businesses and industries with post-graduate researchers. There were poster presentations and talks that lead to exchange of information and productive discussion, which helped us see our research under a different perspective, taking into account different disciplines. |
Year(s) Of Engagement Activity | 2015 |
URL | http://www.biodtp.ox.ac.uk/news/oxford-interdisciplinary-bioscience-networking-event/index.html |
Description | Presentation |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | Presentation and discussion of work at 5th Annual ShanghaiTech BioFrontier held at ShanghaiTech in 20-21 October 2017. |
Year(s) Of Engagement Activity | 2017 |
URL | http://fogg2017.biofrontier.org/ |
Description | Presentation at Cellular Mechanisms in Health and Disease (10-14 November 2019) Shanghai, China |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Presentation at Cellular Mechanisms in Health and Disease (10-14 November 2019) Shanghai, China |
Year(s) Of Engagement Activity | 2019 |
URL | http://bioforum.shanghaitech.edu.cn |
Description | Presentation: Importance of cytoplasmic localisation of ribonucleotide reductase for genome stability |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Importance of cytoplasmic localisation of ribonucleotide reductase for genome stability Christos Andreadis1, Israel Salguero2 and Stephen Kearsey1 1Dept. of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK 2Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP production, and in S.pombe it consists of Cdc22R1 and Suc22R2 subunits. Proper regulation of dNTP levels is crucial for the fidelity of DNA synthesis and repair, and the importance of RNR is reflected in the multiple levels of its regulation. In physiological conditions, Cdc22R1 is predominantly cytoplasmic and Suc22R2 is nuclear, while on DNA damage and in S-phase RNR holoenzyme is active in the cytoplasm. The biological significance of this localisation, away from the DNA synthesis site, is unclear, particularly as studies in mammalian cells suggest that RNR may be targeted to sites of DNA repair. We show that when the colocalization of RNR subunits is prevented, the DNA replication is impeded, albeit the effect on DNA damage sensitivity is little. When both subunits are forced to colocalize in the nucleus, the S-phase is suboptimal and the mutation rate is increased, even though the effect on dNTP levels is minimal. However, in these conditions, rad3? sensitivity to DNA damage-inducing agents is suppressed. Our results indicate that regulation of RNR by subcellular relocalisation of Suc22 is an important but not essential aspect of dNTP control. Furthermore, our results corroborate the evolution of a highly conserved cytoplasmic localization of RNR to optimise the dNTP supply for DNA replication and genome stability. |
Year(s) Of Engagement Activity | 2016 |
URL | http://events.embo.org/16-nucleus/ |
Description | Presentation: Studying potential new mechanisms of ribonucleotide reducatase regulation |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Studying potential new mechanisms of RNR regulation Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the synthesis of deoxyribonucleotides required for DNA synthesis and repair. Inhibition of this enzyme thus blocks cell proliferation, and RNR inhibitors such as hydroxyurea, clorabine and gemciabine are useful in the treatment of some cancers such as leukaemias, pancreatic cancer and ovarian tumours (Shao, 2006). Furthermore, its correct regulation is essential for genome stability (Bester, 2011). The structure of RNR consists of large and small subunits. The small subunit contains a tyrosyl radical cofactor used to generate the thyl radical that is needed for activity of the catalytic site in the large subunit for ribose reduction. RNR is subject to many levels of regulation that include allosteric control, proteolysis, cell cycle regulated expression, sub-cellular localization of subunits and regulation by intrinsically disordered protein (IDPs) inhibitors such Spd1 (reviewed in (Guarino et al, 2014)). Spd1 was identified in fission yeast as a protein inhibiting S phase when overexpressed (Woolard, 1996). In fission yeast, the localisation of large subunit Cdc22 is constitutively pancellular, while the smaller, Suc22, is nuclear, except during S phase of after DNA damage when proteolysis of Spd1 allows the cytoplasmic level of Suc22 to rise. This is thought to activate RNR by allowing interaction of both subunits. However, is known that only the large subunit interacts with Spd1 in vitro (Hakansson, 2006). Although a tetramer of two large and small subunits shows activity, recent structural analysis suggests that the active form of the yeast enzyme may be a holoenzyme consisting of a hexamer comprised by four copies of the large subunit and two of the smaller ((Fairman, 2005), reviewed in (Logan, 2011)). We have discovered that fission yeast cells growing under different conditions show striking foci of Cdc22 that colocalize with Spd1 in the cytoplasm, presumably representing large complexes of the proteins. Previously, it has been reported that Escherichia coli RNR forms inactive R14R24 rings that can interlock to form catenated megacomplexes (Zimanyi, 2012). In addition, it has been described in Saccharomyces cerevisiae that in the presence of DNA damage agents Rnr1 associates with autophagosomal structures (Dyavaiah, 2001). To understand the biological significance of these structures we are studying whether Cdc22 foci are affected by over-expression or deletion of Spd1, or by infliction of DNA damaging agents. Also, we want to know how their frequency of occurrence correlates with activity state of RNR, testing their prevalence in ribonucleotide reductase hyperactive mutants, in quiescent or S phase cells, or whether they are affected by RNR inhibition by hydroxyurea and/or other inhibitors. Furthermore, we have determined that these foci do not colocalize to known organelle-associated structures in the cell,and we are addressing the possibility that the nature of these structures could be related with autophagy processes. |
Year(s) Of Engagement Activity | 2016 |
Description | The joint 26th International Conference on Arginine and Pyrimidines & the 1st International Conference on Aminoacids and Nucleotides, Shanghai, China (4-7 July 2018) (Talk) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Professional Practitioners |
Results and Impact | The joint 26th International Conference on Arginine and Pyrimidines & the 1st International Conference on Aminoacids and Nucleotides, Shanghai, China (4-7 July 2018) (Talk) |
Year(s) Of Engagement Activity | 2018 |
Description | Website on DNA replication |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | Website hits greater than 200 week Feedback from labs wanting to be linked to the website. |
Year(s) Of Engagement Activity | 2013,2014 |
URL | http://www.dnareplication.net |