Duplicated DNA Damage-Inducible Genes DDI2/DDI3 in Budding Yeast Saccharomyces cerevisiae Encodes a Functional Cyanamide Hydratase
Date
2016-11-02
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Doctoral
Abstract
In a yeast genome-wide microarray analysis, two Saccharomyces cerevisiae open reading frames YFL061w and YNL335w displayed the highest fold levels of induction after methyl methanesulfonate (MMS) treatment, a typical DNA damaging agent. Hence the genes were named as DNA damage inducible genes 2 and 3 (DDI2 and DDI3). DDI2 and DDI3 share identical DNA sequences in their coding regions and promoters (except for one nucleotide). Their deduced protein product Ddi2/3 is 36% identical to an identified cyanamide hydratase (EC 4.2.1.69) from a soil fungus Myrothecium verrucaria. The enzyme catalyzes cyanamide hydration to urea. Hence Ddi2/3 likely has the same enzymatic activity. To study the encoded protein, the DDI2/3 open reading frame was cloned into a bacterial expression system, and the resulting recombinant protein was purified for biochemical analysis. The cyanamide hydratase activity of Ddi2/3 was measured by using an enzyme-based urea detection assay. The kinetic study showed that Ddi2/3 has a KM of 17 mM for cyanamide, and kcat is 9.8 ± 0.1 S-1. The cyanamide hydratase activity of endogenous Ddi2/3 was also detected by using yeast whole cell extracts (WCEs).
The cyanamide hydratase domain belongs to the Histidine–Aspartate (HD) domain superfamily. Double mutations in conserved His88 and Asp89 residues abolished the enzymatic activity, suggesting that the conserved HD residues contribute to the cyanamide hydratase activity of Ddi2/3.
To identify whether DDI2/3 can be induced by cyanamide, the DDI2/3 promoter was fused to lacZ as a reporter gene, and the β-galactosidase assay demonstrated that DDI2/3-lacZ can be activated by both MMS and cyanamide, and the optimal fold induction by cyanamide was even higher than by MMS. Interestingly, the basal level expression of DDI2/3-lacZ was extremely low, and yeast WCEs from untreated wild-type cells do not have measurable cyanamide hydratase activity.
To investigate the physiological function of DDI2/3, we constructed yeast ddi2/3Δ single and double null mutant strains by sequential targeted one-step gene disruption via homologous recombination. Disruption of DDI2/3 genes enhanced cellular sensitivity to both MMS and cyanamide by up to 1,000-fold. Furthermore, WCEs from the ddi2/3∆ single mutant had reduced cyanamide hydratase activity, while that from the ddi2Δ ddi3∆ double mutant had no detectable cyanamide hydratase activity. Since the induction of DDI2/3-lacZ by cyanamide and MMS was not compromised in the ddi2/3∆ strains, the DDI2/3 gene is not required for its transcriptional regulation.
Since no structural study with a cyanamide hydratase domain has been reported, we undertook crystallographic studies on Ddi2/3. After protein crystallization, X-ray diffraction, and data interpretation, Ddi2/3 was revealed as a zinc-binding protein and composed of 14 helices A zinc anion is coordinated to three conserved HD residues, H55, H88 and D89, and a water molecule, forming a tetrahedral geometry. To further investigate the active site, several amino acid substitutions were made, and enzymatic assays revealed that H137, Q138, T157, and N161 are required for the activity.
HD domain represents a superfamily of metal-dependent phosphohydrolases and characterized members are involved in nucleotide metabolism and signal transduction. However, CAH has been reported as an exception. Hence, the study on Ddi2/3 will enrich our knowledge of the variation of HD domain proteins, and has identified a unique subfamily within the HD domain family.
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Keywords
DDI2/DDI3, Saccharomyces cerevisiae, cyanamide hydratase
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Microbiology and Immunology
Program
Microbiology and Immunology