Functions and transcriptional regulation of the budding yeast DDI2 gene

DDI 2和DDI 3是通过我们的微阵列分析鉴定的两个DNA损伤诱导基因。这两个基因的开放阅读框和启动子序列相同，随后称为DDI 2基因。与大多数其他特征良好的DDI基因不同，DDI 2仅由选定的DNA烷基化剂高度诱导，而不是由辐射或其他类型的遗传毒性化学物质诱导。Ddi 2蛋白的序列分析表明，它可能编码一个含有HD结构域的氨腈水合酶（CAH）。事实上，纯化的重组Ddi 2显示出真正的CAH活性，更有趣的是，Ddi 2基因被氰胺（CY）诱导高达20，000倍，并且Ddi 2和Ddi 3基因的缺失使细胞对CY和DNA损伤剂甲磺酸甲酯（MMS）敏感。在这里，我们建议进一步描述这对新的基因。目的1：研究Ddi 2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Ddi 2蛋白，并获得了初步晶体。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，确定这种酶的天然底物和详细的反应机制。此外，还将研究Ddi 2对CY解毒的生物化学作用及其可能参与DNA损伤反应。目的二是探讨DDI 2诱导的信号转导机制。我们已经证明Ddi 2不参与其自身的转录调控，但发现转录激活因子Fzf 1是CY和MMS诱导Ddi 2所必需的。我们将进行全基因组筛选，以寻找具有改变的DDI 2诱导谱的酵母缺失突变体。该筛选将允许我们鉴定和表征响应于CY和MMS处理而导致DDI 2诱导的CY受体和信号转导子。然后将建立信号转导级联。**目的3是通过利用DDI 2调节系统开发新的实验工具。这些试剂有望上级现有的芽殖酵母诱导表达系统，并将有利于研究和工业界。一旦DDI 2调控机制被阐明，该表达系统就有可能适用于高等真核生物，包括植物和哺乳动物。目的4是研究一个新的观察结果背后的分子机制，DDI 2和DDI 3基因的缺失导致氮饥饿期间短暂的过度生长。我们计划测试一个假设，Ddi 2作为一个G1-S细胞周期检查点，通过传感氮的可用性。更具体地，我们提出Ddi 2是Cln 3-Cdc 28和/或Bck 2的负调节剂。预计这项拟议的研究项目将揭示一个很大程度上未表征的基因，其功能和调控。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.