Response of the Green Alga Chlamydomonas reinhardtii to the DNA Damaging Agent Zeocin

Čížková, Mária; Slavková, Monika; Vítová, Milada; Zachleder, Vilém; Bišová, Kateřina

doi:10.3390/cells8070735

Cited by 16 publications

(6 citation statements)

References 67 publications

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“…We first confirmed that inactivation of KU80 , KU70 , and POLQ caused the expected sensitivity to the drug Zc which creates DSBs ( Ehrenfeld et al 1987 ; Chankova et al 2007 ; Chen et al 2008 ; Čížková et al 2019 ). Surprisingly, polQ cells were found to be more sensitive to Zc than ku80 and ku70 cells.…”

Section: Resultssupporting

confidence: 55%

Chlamydomonas POLQ is necessary for CRISPR/Cas9-mediated gene targeting

Сизова

Kelterborn

Verbenko

et al. 2021

G3 Genes|Genomes|Genetics

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The use of CRISPR/Cas endonucleases has revolutionized gene editing techniques for research on Chlamydomonas reinhardtii. To better utilize the CRISPR/Cas system, it is essential to develop a more comprehensive understanding of the DNA repair pathways involved in genome editing. In this study, we have analyzed contributions from canonical KU80/KU70-dependent non-homologous end-joining and polymerase theta (POLQ)-mediated end-joining on SpCas9-mediated untemplated mutagenesis and homology-directed repair/gene inactivation in Chlamydomonas. Using CRISPR/SpCas9 technology, we generated DNA repair-defective mutants ku80, ku70, polQ for gene targeting experiments. Our results show that untemplated repair of SpCas9-induced double strand breaks results in mutation spectra consistent with an involvement of both KU80/KU70 and POLQ. In addition, the inactivation of POLQ was found to negatively affect homology-directed repair of the inactivated paromomycin resistant mut-aphVIII gene when donor single-stranded oligos were used. Nevertheless, mut-aphVIII was still repaired by homologous recombination in these mutants. POLQ inactivation suppressed random integration of transgenes co-transformed with the donor ssDNA. KU80 deficiency did not affect these events but instead was surprisingly found to stimulate homology-directed repair/gene inactivation. Our data suggests that in Chlamydomonas, POLQ is the main contributor to CRISPR/Cas-induced homology-directed repair and random integration of transgenes, while KU80/KU70 potentially plays a secondary role. We expect our results will lead to improvement of genome editing in Chlamydomonas reinhardtii and can be used for future development of algal biotechnology.

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Section: Resultssupporting

confidence: 55%

Chlamydomonas POLQ is necessary for CRISPR/Cas9-mediated gene targeting

Сизова

Kelterborn

Verbenko

et al. 2021

G3 Genes|Genomes|Genetics

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“…There were also some phenotypic markers that overlapped with certain chemicals. For example, cells treated with H 2 O 2 and zeocin, a DNA damaging agent, 28 both garnered the same top three chloroplast normalized moment features. However, the VIP ranking order of these phenotypic markers and the overall combination of markers were still distinct between chemical profiles.…”

Section: ■ Results and Discussionmentioning

confidence: 94%

Using a Novel Multiplexed Algal Cytological Imaging (MACI) Assay and Machine Learning as a Way to Characterize Complex Phenotypes in Plant-Type Organisms

Ostovich,

Klaper

2024

Environ. Sci. Technol.

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High-throughput phenotypic profiling assays, popular for their ability to characterize alternations in single-cell morphological feature data, have been useful in recent years for predicting cellular targets and mechanisms of action (MoAs) for different chemicals and novel drugs. However, this approach has not been extensively used in environmental toxicology due to the lack of studies and established methods for performing this kind of assay in environmentally relevant species. Here, we developed a multiplexed algal cytological imaging (MACI) assay, based on the subcellular structures of the unicellular microalgae, Raphidocelis subcapitata, a toxicology and ecological model species. Several different herbicides and antibiotics with unique MoAs were exposed to R. subcapitata cells, and MACI was used to characterize cellular impacts by measuring subtle changes in their morphological features, including metrics of area, shape, quantity, fluorescence intensity, and granularity of individual subcellular components. This study demonstrates that MACI offers a quick and effective framework for characterizing complex phenotypic responses to environmental chemicals that can be used for determining their MoAs and identifying their cellular targets in plant-type organisms.

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“…There is a scarcity of data [ 40 ] indicating that H 2 O 2 and NO can modify algal cell development by influencing the expression of cell cycle-related proteins, such as cyclin and cyclin-dependent kinases and histone proteins [ 5 , 41 ]. These examples form a basis for establishing the role of ROS and RNS in the cell cycle of green algae, both as the signals directly involved in the regulation of protein expression required for completing the cell cycle, and as factors involved in the regulation of photoautotrophic growth [ 42 ], which, in turn, allow the cell to attain the commitment competence.…”

Section: H 2 O 2 and No Cross-talkmentioning

confidence: 99%

Cross Talk between Hydrogen Peroxide and Nitric Oxide in the Unicellular Green Algae Cell Cycle: How Does It Work?

Pokora

Tułodziecki

Dettlaff-Pokora

et al. 2022

Cells

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The regulatory role of some reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as hydrogen peroxide or nitric oxide, has been demonstrated in some higher plants and algae. Their involvement in regulation of the organism, tissue and single cell development can also be seen in many animals. In green cells, the redox potential is an important photosynthesis regulatory factor that may lead to an increase or decrease in growth rate. ROS and RNS are important signals involved in the regulation of photoautotrophic growth that, in turn, allow the cell to attain the commitment competence. Both hydrogen peroxide and nitric oxide are directly involved in algal cell development as the signals that regulate expression of proteins required for completing the cell cycle, such as cyclins and cyclin-dependent kinases, or histone proteins and E2F complex proteins. Such regulation seems to relate to the direct interaction of these signaling molecules with the redox-sensitive transcription factors, but also with regulation of signaling pathways including MAPK, G-protein and calmodulin-dependent pathways. In this paper, we aim to elucidate the involvement of hydrogen peroxide and nitric oxide in algal cell cycle regulation, considering the role of these molecules in higher plants. We also evaluate the commercial applicability of this knowledge. The creation of a simple tool, such as a precisely established modification of hydrogen peroxide and/or nitric oxide at the cellular level, leading to changes in the ROS-RNS cross-talk network, can be used for the optimization of the efficiency of algal cell growth and may be especially important in the context of increasing the role of algal biomass in science and industry. It could be a part of an important scientific challenge that biotechnology is currently focused on.

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Response of the Green Alga Chlamydomonas reinhardtii to the DNA Damaging Agent Zeocin

Cited by 16 publications

References 67 publications

Chlamydomonas POLQ is necessary for CRISPR/Cas9-mediated gene targeting

Chlamydomonas POLQ is necessary for CRISPR/Cas9-mediated gene targeting

Using a Novel Multiplexed Algal Cytological Imaging (MACI) Assay and Machine Learning as a Way to Characterize Complex Phenotypes in Plant-Type Organisms

Cross Talk between Hydrogen Peroxide and Nitric Oxide in the Unicellular Green Algae Cell Cycle: How Does It Work?

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