This project is concerned with the structural and functional aspects of a dGTPase of the bacterium E. coli, which has the unusual specificity: dGTP->dG + PPPi. Although this activity was reported a number of years ago, its function in the cell has been unknown. Recently, we discovered a novel mutator activity in E. coli resulting from a defect in the dgt gene, encoding the above activity. Thus, insight into the function of the Dgt dGTPase may be gleaned from studies of the dgt mutator effect. As a primary hypothesis, we are considering the possibility that Dgt may be an activity aimed at sanitizing the cellular dNTP pool by removing aberrant dGTP derivatives. These derivatives, if not removed, will be incorporated into DNA and cause mutations. Our studies of the putative Dgt mutation prevention mechanism include several approaches: (i) a genetic analysis of the dgt mutator effect in the bacterium E. coli, (ii) a biochemical analysis of the Dgt protein, including study of its substrate specificity, and (iii) analysis of Dgt protein by Nuclear Magnetic Resonance and X-ray crystallography methods. NMR studies will be aimed at possibly identifying the physiologically relevant substrate. It is noted that other mutation avoidance pathways working at the dNTP level, such as hydrolysis of the oxidative stress-related contaminant 8-oxo-dGTP, are characterized by a more conventional biochemical transformation in which the pyrophosphate moiety is cleaved rather than the entire triphosphate chain. Thus, it is likely that further study of the Dgt enzyme will provide insight into a novel, and likely important, cellular mechanism of DNA damage and mutation avoidance, whose significance may likely extend beyond the E. coli model system.

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Itsko, Mark; Schaaper, Roel M (2017) Suppressors of dGTP Starvation in Escherichia coli. J Bacteriol 199:
Itsko, Mark; Schaaper, Roel M (2016) Transcriptome Analysis of Escherichia coli during dGTP Starvation. J Bacteriol 198:1631-44
Singh, Deepa; Schaaper, Roel M; Hochkoeppler, Alejandro (2016) A continuous spectrophotometric enzyme-coupled assay for deoxynucleoside triphosphate triphosphohydrolases. Anal Biochem 496:43-9
Singh, Deepa; Gawel, Damian; Itsko, Mark et al. (2015) Structure of Escherichia coli dGTP triphosphohydrolase: a hexameric enzyme with DNA effector molecules. J Biol Chem 290:10418-29
Itsko, Mark; Schaaper, Roel M (2014) dGTP starvation in Escherichia coli provides new insights into the thymineless-death phenomenon. PLoS Genet 10:e1004310
Itsko, Mark; Schaaper, Roel M (2011) The dgt gene of Escherichia coli facilitates thymine utilization in thymine-requiring strains. Mol Microbiol 81:1221-32
Ye, Wenjie; Sangaiah, R; Degen, Diana E et al. (2009) Iminohydantoin lesion induced in DNA by peracids and other epoxidizing oxidants. J Am Chem Soc 131:6114-23
Gawel, Damian; Hamilton, Michael D; Schaaper, Roel M (2008) A novel mutator of Escherichia coli carrying a defect in the dgt gene, encoding a dGTP triphosphohydrolase. J Bacteriol 190:6931-9