Bibliographic Details
| Title: |
Sensing chemical-induced DNA damage using CRISPR/Cas9-mediated gene-deletion yeast-reporter strains. |
| Authors: |
Yamamoto, Kosuke1 (AUTHOR), Tochikawa, Shintaro1 (AUTHOR), Miura, Yuuki1 (AUTHOR), Matsunobu, Shogo1 (AUTHOR), Hirose, Yuu1,2 (AUTHOR), Eki, Toshihiko1 (AUTHOR) eki@chem.tut.ac.jp |
| Source: |
Applied Microbiology & Biotechnology. 2/1/2024, Vol. 108 Issue 1, p1-17. 17p. |
| Subject Terms: |
*GENETIC toxicology, *DNA damage, *DNA repair, *EXCISION repair, *CRISPRS, *METHYL methanesulfonate |
| Abstract: |
Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated and single-copy plasmid–based reporter assays in budding yeast using a RNR3 promoter–driven luciferase gene. These assays were designed to compare the response to genotoxic chemicals with a pre-established multicopy plasmid–based assay. Despite exhibiting the lowest luciferase activity, the chromosomally integrated reporter assay showed the highest fold induction (i.e., the ratio of luciferase activity in the presence and absence of the chemical) compared with the established plasmid-based assay. Using CRISPR/Cas9 technology, we generated mutants with single- or double-gene deletions, affecting major DNA repair pathways or cell permeability. This enabled us to evaluate reporter gene responses to genotoxicants in a single-copy plasmid–based assay. Elevated background activities were observed in several mutants, such as mag1Δ cells, even without exposure to chemicals. However, substantial luciferase induction was detected in single-deletion mutants following exposure to specific chemicals, including mag1Δ, mms2Δ, and rad59Δ cells treated with methyl methanesulfonate; rad59Δ cells exposed to camptothecin; and mms2Δ and rad10Δ cells treated with mitomycin C (MMC) and cisplatin (CDDP). Notably, mms2Δ/rad10Δ cells treated with MMC or CDDP exhibited significantly enhanced luciferase induction compared with the parent single-deletion mutants, suggesting that postreplication and for nucleotide excision repair processes predominantly contribute to repairing DNA crosslinks. Overall, our findings demonstrate the utility of yeast-based reporter assays employing strains with multiple-deletion mutations in DNA repair genes. These assays serve as valuable tools for investigating DNA repair mechanisms and assessing chemical-induced DNA damage. Key points: • Responses to genotoxic chemicals were investigated in three types of reporter yeast. • Yeast strains with single- and double-deletions of DNA repair genes were tested. • Two DNA repair pathways predominantly contributed to DNA crosslink repair in yeast. [ABSTRACT FROM AUTHOR] |
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