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

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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.