STRUCTURAL STUDIES OF EUKARYOTIC RIBONUCLEOTIDE REDUCTASE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 核糖核苷酸还原酶（RNR）通过将NDP还原为dNDP来催化从头DNA合成的限速步骤。虽然存在许多关于真核RNR非催化亚基的结构和生化数据，特别是小鼠Rnr 2和酵母Rnr 2 p？Rnr 4p，到目前为止还没有任何真核生物Rnr 1的结构报道。使用BioCARS的MAD数据，我们解决了酵母Rnr 1结构。在此建议中，我们将解决几个同源效应子-底物复合物结构，以解决底物如何通过效应子位点的特异性dNTPs结合来选择。酵母RNR组装涉及Rnr 2-Rnr 4与Rnr 1的缔合。Rnr 2和Rnr 4的C-末端肽可以破坏这种组装，并且基于Rnr 2肽设计了一类新的抗癌和抗病毒抑制剂。我们将解决与拟肽库和抗癌药物如氯法拉滨复合的Rnr 1的结构。在酵母中，RNR活性由ATP（上调剂）和dATP（下调剂）变构控制。我们将解决与ATP和dATP复合的Rnr 1的结构来解决这个问题。酵母RNR也被Sml 1下调，Sml 1结合Rnr 1。Sml 1的C-末端抑制RNR活性，但效力降低。我们将使用MAD和与Sml 1衍生肽和完整Sml 1复合的Rnr 1解析Sml 1结构。我们构建了大量的Rnr 1突变体来研究其结构与功能的关系。有几个已经结晶的天然形式和复杂的效应-底物复合物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step of de novo DNA synthesis by reducing NDPs to dNDPs. Though much structural and biochemical data on non-catalytic subunits of eukaryotic RNR exist, specifically mouse Rnr2, and yeast Rnr2p?Rnr4p, until now there is no reported structure for any eukaryotic Rnr1. Using MAD data from BioCARS, we solved the yeast Rnr1 structure. During this proposal we will solve several cognate effector-substrate complex structures to address how substrates are selected by specific dNTPs binding at the effector site. The yeast RNR assembly involves association of Rnr2-Rnr4 with Rnr1. C-terminal peptides of Rnr2 and Rnr4 can disrupt this assembly, and a new class of anticancer and antiviral inhibitors is designed based on Rnr2 peptides. We will solve structures of Rnr1 complexed with peptidomimetic libraries and anticancer agents like clofarabine. In yeast RNR activity is controlled allosterically by ATP (upregulator) and dATP (downregulator). We will solve the structures of Rnr1 complexed with ATP and dATP to address this. Yeast RNR is also downregulated by Sml1, which binds Rnr1. The C-termini of Sml1 inhibits RNR activity with reduced potency. We will solve the Sml1 structure using MAD and Rnr1 complexed with Sml1-derived peptides and intact Sml1. We have constructed numerous mutants of Rnr1to study structure-function relationships. Several have been crystallized both in the native form and in complex with effector-substrate complexes.