Functions and transcriptional regulation of the budding yeast DDI2 gene

DDI2和DDI3是通过我们的微阵列分析确定的两个DNA损伤诱导基因。这两个基因在其开放阅读框架和启动子序列上是相同的，随后被称为DDI2基因。与大多数其他特征良好的DDI基因不同，DDI2只被选定的DNA烷基化试剂高度诱导，而不是辐射或其他类型的遗传毒性化学物质。序列分析表明，Ddi2蛋白可能编码一个含有氰胺水合酶(CAH)的HD结构域。事实上，纯化的重组Ddi2显示出真正的CAH活性，更有趣的是，DDI2基因被氰胺(CY)诱导了高达20,000倍，DDI2和DDI3基因的缺失使细胞对CY和DNA损伤剂甲烷磺酸甲酯(MMS)都敏感。在这里，我们建议进一步鉴定这对新基因。**目的1是鉴定Ddi2蛋白的结构和酶活性。为了实现上述目标，我们制备了大量纯化的重组Ddi2蛋白，并获得了初步的晶体。我们将通过X射线结晶学来确定蛋白质的结构，并通过各种手段，确定这种酶的天然底物(S)和详细的反应机理。此外，还将探讨Ddi2对CY解毒的生化作用及其可能参与的DNA损伤反应。**目的2是探讨导致DDI2诱导的信号转导的分子机制。我们已经证明Ddi2不参与其自身的转录调控，但发现转录激活因子Fzf1是CY和MMS诱导DDI2所必需的。我们将进行全基因组筛选，寻找具有改变的DDI2诱导谱的酵母缺失突变体。这一筛选将使我们能够识别和表征CY受体和信号转导蛋白(S)，它们导致在CY和MMS处理后诱导DDI2的产生。然后将建立信号转导级联。**目标3是利用DDI2调节系统开发新的实验工具。这些试剂有望在芽殖酵母中优于现有的诱导表达系统，并将有益于研究和工业社区。一旦DDI2的调控机制被阐明，该表达系统就有可能适应于包括植物和哺乳动物在内的高等真核生物。**目的4是研究在氮饥饿期间DDI2和DDI3基因的缺失导致瞬时过度生长的分子机制。我们计划通过感知氮的可获得性来检验Ddi2作为G1-S细胞周期检查点的假设。更具体地说，我们认为Ddi2是Cln3-CDC28和/或Back2的负调控因子。**预计这项拟议的研究项目将揭示一个基本上未描述的基因、其功能和调节。鉴于CY在农业和公共卫生方面的经济重要性，拟议中的研究也将造福于我们的生活。

DDI2 and DDI3 are two DNA-damage inducible genes identified through our microarray analysis. The two genes are identical in their open reading frame and promoter sequences, and are subsequently referred to as DDI2 gene. Differing from most other well-characterized DDI genes, DDI2 is only highly induced by selected DNA-alkylating agents but not by radiations or other types of genotoxic chemicals. Sequence analysis of the deduced Ddi2 protein indicates that it may encode an HD-domain containing cyanamide hydratase (CAH). Indeed, purified recombinant Ddi2 displays bona fide CAH activity and more interestingly, the DDI2 gene is induced by cyanamide (CY) by up to 20,000-fold, and deletion of both DDI2 and DDI3 genes sensitizes cells to both CY and the DNA-damaging agent methyl methanesulfonate (MMS). Here we propose to further characterize this novel pair of genes.**Aim 1 is to characterize the Ddi2 protein structure and enzymatic activity. Toward the above goals, we have made large quantities of purified recombinant Ddi2 protein and obtained preliminary crystals. We will determine the protein structure by X-ray crystallography and through a variety of means, determine the native substrate(s) for this enzyme and the detailed mechanism of reaction. In addition, the biochemical roles of CY detoxification by Ddi2 and its possible involvement in DNA damage response will also be investigated.**Aim 2 is to interrogate the molecular mechanism of signal transduction leading to DDI2 induction. We have demonstrated that Ddi2 is not involved in its own transcriptional regulation, but found that the transcriptional activator Fzf1 is absolutely required for the induction of DDI2 by CY and MMS. We will perform a genome-wide screen to look for yeast deletion mutants with altered DDI2 induction profile. This screen will allow us to identify and characterize CY receptor and signal transducer(s) that lead to the induction of DDI2 in response to CY and MMS treatment. The signal transduction cascade will then be established.**Aim 3 is to develop novel experimental tools by utilizing the DDI2 regulatory system. These reagents are expected to be superior to the existing inducible expression systems in budding yeast and will benefit the research and industrial community. Once the DDI2 regulatory mechanism is elucidated, there is potential that the expression system can be adapted to higher eukaryotes including plants and mammals.**Aim 4 is to investigate the molecular mechanism behind a novel observation that deletion of both DDI2 and DDI3 genes result in a transient overgrowth during nitrogen starvation. We plan to test a hypothesis that Ddi2 acts as a G1-S cell cycle checkpoint by sensing nitrogen availability. More specifically, we propose that Ddi2 is a negative regulator of the Cln3-Cdc28 and/or Bck2.**It is expected that this proposed research project will uncover a largely uncharacterized gene, its functions and regulation. Given the economic importance of CY in agriculture and public health, the proposed research will also benefit our life.