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Description

E.coli possess a unique enzyme, deoxyguanosine triphosphohydrolase (dGTPase) whichhydrolyzes dGTP into deoxyguanosine (dG) and triphosphate. This enzyme, a homologue of human SAMHD1, is thought to play a role in DNA fidelity because the deletion of its encoding gene, dgt, has a mutator effect. Interestingly, a mutant dGTPase enzyme (M9) was found to be more active than the wild-type enzyme and when M9 overexpression was induced in E. coli strain BL21-AI, a few hours post-induction severe cell death was observed. We hypothesize that overexpression of M9 results in dGTP starvation, which decreases the intracellular dGTP concentration. To investigate this toxic effect, cells were stained with DNA specific fluorescent dyes, DAPI and Hoechst, and visualized using confocal microscopy. Results indicate induced cells exhibited increased size and reduced DNA compaction. The nucleoid of the cell was compromised, and cells lost their DNA. TUNEL assays showed an increase of DNA breaks in induced cells. Overall, results suggest that induction of M9 leads to DNA degradation and loss of integrity potentially contributing to cell death. Understanding this mechanism could aid in advancing our knowledge on the impacts of mutations on genome fidelity and could also be exploited for an antibiotic alternative in the context of rising antibiotic resistance. Further investigation is required to understand how induced dGTP starvation results in cell death.

Publication Date

4-1-2025

Keywords

dGTPase, E. coli, M9 mutant, overexpression, dGTP starvation, DNA degradation, cell death, genome integrity, confocal microscopy, DAPI staining, Hoechst staining, TUNEL assay, mutator effect, SAMHD1 homologue, antibiotic resistance, DNA replication stress, nucleotide pool imbalance, SOS response, purine metabolism, synthetic lethality

Investigating The Cell Killing Mechanism During dGTP Starvation

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