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复制遗传物质DNA。DNA的复制需要dNTP构建块，这些构建块不是染色体复制所必需的，但也参与了其他染色体过程，如DNA修复和重组。DNTPs产生的一个重要步骤涉及一种名为核糖核苷酸还原酶的酶，该酶催化该途径中的限速步骤。必须启动核糖核苷酸还原酶才能进行染色体复制，因为细胞中的游离dNTPs池只够复制一小部分基因组。同样重要的是，不要一直打开核糖核苷酸还原酶，因为高浓度的dNTPs会降低复制DNA的DNA聚合酶的保真度，从而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，抑制该酶的药物在医学上很有用，因为它们可以通过抑制DNA复制来减缓细胞的增殖，这可以用于癌症和其他有利于减缓细胞分裂的疾病的治疗。核糖核苷酸还原酶在细胞中以几种方式调节，反映了这一过程的重要性。一种机制涉及与核糖核苷酸还原酶结合的小蛋白抑制剂。到目前为止，这些蛋白质抑制剂只在酵母中发现，但有充分的理由预计它们也在人类细胞中发挥作用。在这项资助中，我们将研究一种特殊的核糖核苷酸还原酶抑制剂Spd1，它是在分裂酵母中发现的。虽然已知Spd1抑制核糖核苷酸还原酶，但它的作用方式还不是很清楚。我们将使用一些方法来研究Spd1抑制的机制，这些方法可以用来检测活细胞中Spd1与核糖核苷酸还原酶亚单位之间的相互作用。利用酵母遗传学，我们继续选择耐Spd1抑制的核糖核苷酸还原酶突变体，对这些突变的分析应该有助于阐明Spd1抑制的机制。具体地说，我们可以观察这些突变发生在RNR结构中的哪里，这可能有助于定义Spd1在酶上的结合表面。核糖核苷酸还原酶在进化中高度保守，我们将探索Spd1是否能抑制多细胞生物细胞中的核糖核苷酸还原酶，这将是确定类似的蛋白抑制物在人类细胞中是否发挥作用的一步。了解这种抑制机制可能是有用的，因为它可能会提出新的方法来开发抑制核糖核苷酸还原酶的方法，从而扩大目前可用的药物范围。

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.