Two duplicated genes DDI2 and DDI3 in budding yeast encode a cyanamide hydratase and are induced by cyanamide.

Two DNA damage-inducible genes in Saccharomyces cerevisiae, DDI2 and DDI3, are identical and encode putative HD domain-containing proteins, whose functions are currently unknown. Because Ddi2/3 also shows limited homology to a fungal cyanamide hydratase that converts cyanamide to urea, we tested the enzymatic activity of recombinant Ddi2. To this end, we developed a novel enzymatic assay and determined that the Km value of the recombinant Ddi2/3 for cyanamide is 17.3 ± 0.05 mm, and its activity requires conserved residues in the HD domain. Unlike most other DNA damage-inducible genes, DDI2/3 is only induced by a specific set of alkylating agents and surprisingly is strongly induced by cyanamide. To characterize the biological function of DDI2/3, we sequentially deleted both DDI genes and found that the double mutant was unable to metabolize cyanamide and became much more sensitive to growth inhibition by cyanamide, suggesting that the DDI2/3 genes protect host cells from cyanamide toxicity. Despite the physiological relevance of the cyanamide induction, DDI2/3 is not involved in its own transcriptional regulation. The significance of cyanamide hydratase activity and its induced expression is discussed.

Isolation of yeast nucleic acids.

Saccharomyces cerevisiae is a well-established model organism used to study multiple facets of eukaryotic organisms. The manipulation and isolation of DNA is a key element of basic genetic research. Meanwhile, the isolation of RNA is required for the study of transcriptional regulation. Presented in this chapter are fast and efficient methods of isolating genomic and plasmid DNA and total RNA that is capable of being utilized for a variety of genetic studies such as restriction analysis, northern and southern blotting, and real-time reverse-transcriptase PCR. Plasmids isolated via this method are also of sufficient quality to be transformed into E. coli for further genetic manipulation and study.

Selective tumor killing based on specific DNA-damage response deficiencies.

Organisms constantly undergo various stresses within their life span, which can damage their DNA. In order to maintain genomic stability and counteract the development of unwanted genomic mutations, organisms have evolved a DNA-damage response (DDR) to protect their genome. Due to the critical roles played by DDR in genomic stability, its defects can lead to cellular transformation and potentially tumorigenesis. Consequently, this also provides the opportunity to specifically target tumor cells due to a weakened ability to tolerate genotoxic stresses. In this lies a treatment strategy in which the inhibition of remaining DDR pathways can hyper-sensitize tumors to chemotherapeutic agents while minimizing deleterious effects to healthy cells. Therefore it is important to understand the genotypic background of specific tumors to determine which DDR pathways remain and can be targeted for inhibition. Tumor therapies based on the DDR are ideal not only as a means of increasing the effectiveness of current chemotherapies but also as a means to selectively target tumor cells while leaving healthy cells unharmed. Thus, targeting DDR components as a means of increasing effectiveness and discrimination of current chemotherapeutic tumor treatments is currently the focus of many studies and clinical trials.