Functions and transcriptional regulation of the budding yeast DDI2 gene

DDI2和DDI3是通过微阵列分析鉴定的两个dna损伤诱导基因。这两个基因在开放阅读框和启动子序列上是相同的，随后被称为DDI2基因。与大多数其他特征明确的DDI基因不同，DDI2仅由选定的dna烷基化剂高度诱导，而不受辐射或其他类型的遗传毒性化学物质的诱导。Ddi2蛋白的序列分析表明，它可能编码含有hd结构域的氰酰胺水合酶（CAH）。事实上，纯化的重组Ddi2显示出真正的CAH活性，更有趣的是，Ddi2基因被氰酰胺（CY）诱导高达20,000倍，Ddi2和DDI3基因的缺失使细胞对CY和dna损伤剂甲基甲烷磺酸盐（MMS）敏感。在这里，我们建议进一步表征这对新的基因。**目的1是表征Ddi2蛋白结构和酶活性。为此，我们制备了大量纯化的重组Ddi2蛋白，并获得了初步结晶。我们将通过x射线晶体学确定蛋白质结构，并通过各种手段确定该酶的天然底物和反应的详细机制。此外，还将研究Ddi2解毒CY的生化作用及其可能参与的DNA损伤反应。**目的2是探究导致DDI2诱导的信号转导分子机制。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所绝对需要的。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。该筛选将使我们能够识别和表征CY受体和信号换能器，这些受体和信号换能器在CY和MMS处理下诱导DDI2。然后建立信号转导级联。**目标3是利用DDI2调控系统开发新的实验工具。这些试剂有望优于现有的芽殖酵母诱导表达系统，并将使研究和工业界受益。一旦DDI2调控机制被阐明，该表达系统有可能适用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DDI2和DDI3基因缺失导致氮饥饿期间短暂过度生长的分子机制。我们计划通过感知氮的可用性来验证Ddi2作为G1-S细胞周期检查点的假设。更具体地说，我们提出Ddi2是Cln3-Cdc28和/或Bck2的负调控因子。**期望这项研究项目将揭示一个很大程度上未被描述的基因，它的功能和调控。鉴于CY在农业和公共卫生方面的经济重要性，拟议的研究也将有益于我们的生活。

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.