Grant: Discovery Grants Program - Individual

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.