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

细胞分裂所需的一个基本步骤是遗传物质DNA的复制。DNA的复制需要dNTP构建块，这些不是染色体复制所需的，但也涉及其他染色体过程，如DNA修复和重组。dNTPs生产中的一个重要步骤涉及一种称为核糖核苷酸还原酶的酶，该酶催化该途径中的限速步骤。核糖核苷酸还原酶必须被开启以允许染色体复制，因为细胞中的游离dNTPs池仅足以复制基因组的一小部分。同样重要的是，核糖核苷酸还原酶不是一直开启的，因为高浓度的dNTP降低了复制DNA的DNA聚合酶的保真度，产生对细胞有害的突变。由于核糖核苷酸还原酶具有如此关键的作用，抑制这种酶的药物在医学上是有用的，因为通过抑制DNA复制，它们可以减缓细胞增殖，这可以用于治疗癌症和其他疾病，其中减缓细胞分裂是有益的。核糖核苷酸还原酶在细胞中以多种方式调节，反映了该过程的重要性。一种机制涉及与核糖核苷酸还原酶结合的小蛋白抑制剂。这些蛋白质抑制剂迄今只在酵母中发现，但有充分的理由期待它们在人类细胞中也发挥作用。在这项资助中，我们将研究一种在裂变酵母中发现的核糖核苷酸还原酶特异性抑制剂Spd 1。虽然已知Spd 1抑制核糖核苷酸还原酶，但其作用方式尚不清楚。我们将使用可用于检测活细胞中Spd 1与核糖核苷酸还原酶亚基之间相互作用的方法来研究Spd 1抑制的机制。使用酵母遗传学，我们继续选择对Spd 1抑制有抗性的核糖核苷酸还原酶突变体，对这些突变体的分析应该有助于阐明Spd 1抑制的机制。具体来说，我们可以看看这些突变在RNR结构中发生的位置，这可能有助于确定酶上Spd 1的结合表面。核糖核苷酸还原酶在进化中高度保守，我们将探索Spd 1是否可以抑制来自多细胞生物细胞的核糖核苷酸还原酶，这将是确定类似蛋白质抑制剂是否在人类细胞中发挥作用的一步。了解这种抑制机制可能是实际有用的，因为它可能会建议开发抑制核糖核苷酸还原酶的新方法，从而扩大目前可用药物的范围。

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.