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, the function of the Dgt dGTPase may be related to aspects of mutation prevention. In particular, we are considering the possibility that Dgt may be an activity aimed at sanitizing the cellular dNTP pool by removing aberrant dGTP derivatives, which, if not removed, may be incorporated into DNA and cause mutations. We are engaged in two parallel approaches: 1) A genetic analysis is performed in E.
coli aim ed at determining the mutational specificity of the novel mutator and its further genetic requirements. 2) We are purifying Dgt protein for the purpose of analyzing its structure and possible reaction mechanism by Nuclear Magnetic Resonance and X-ray crystallography methods. In particular, NMR studies will be performed on the enzyme in order to identify the physiologically relevant substrate. These include structural studies of isotopically labeled dGTPase, as well as transferred NOE studies on various nucleoside and nucleotides in the presence of the enzyme. Initial efforts have focused on improving the yield of the enzyme prior to isotopic labeling, since the cost of labeled media is significant. It is noted that other mutation avoidance pathways that have been described working at the dNTP level, e.g., the hydrolysis of dNTP pool contaminants 8-oxo-dGTP or 2-hydroxy-dATP, proceed through the well-known MutT-class enzymes which hydrolyze dNTPs according to the reaction: dNTP -> dNMP + PPi. 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.
| 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 (2011) The dgt gene of Escherichia coli facilitates thymine utilization in thymine-requiring strains. Mol Microbiol 81:1221-32 |
