Understanding how ribonucleotide reductase is regulated by the intrinsically disordered protein Spd1

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 (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.

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