What’s broken needs fixing: In-vivo analysis of DNA repair in plants using microirradiation and time-lapse imaging techniques

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14740%2F25%3A00143971" target="_blank" >RIV/00216224:14740/25:00143971 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.efbiotechnology.org/g4g" target="_blank" >https://www.efbiotechnology.org/g4g</a>

DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

What’s broken needs fixing: In-vivo analysis of DNA repair in plants using microirradiation and time-lapse imaging techniques

Popis výsledku v původním jazyce

While we know that crucial elements of DNA repair pathways are evolutionary conserved from plants to humans, relatively little is known about the precise timing of the different DNA repair steps. DNA lesions need to be first recognized by dedicated protein complexes (e.g. the MRE11-RAD50-NBS1; MRN complex), the chromatin structure subsequently remodelled to allow access for repair enzymes, and finally the gaps need to be sealed and the chromatin structure restored. This whole processed can be mapped using biochemical studies and gene expression studies (e.g. gene upregulation after treatment with genotoxic agents). Laser microirradiation, first introduced in mammalian cell lines (cit, cit) is a technique that uses high power lasers to induce DNA damage in a defined cellular region using a confocal microscopy system, offering the possibility to study DNA repair in-vivo in unperturbed cells and has been routinely used in cell lines to study the function of different proteins in DNA repair. We have recently adapted this technique for use in plant systems (Nespor-Dadejova et al., 2022), which required several optimization steps that related to obstacles such as tissue thickness, light scattering and fragility of plant protoplasts. We have shown that the recruitment of factors such as the DNA clamp PCNA, and recognition factors such as MRE11 or PARP1 occurs in the matter of seconds after damage induction in plant systems, with dynamic recruitment to the sites of DNA lesions as evidenced by fluorescence recovery after photobleaching. In a new chapter of this research, we study DNA repair in the context of plant tissues (e.g. seedling roots), where we monitor locally damaged roots in a large temporal window, studying when and if the damaged cells are able to recover. Our first results indicate surprisingly dynamic behavior of the damaged sites. Importantly, we establish a system for root imaging that does not interfere with cellular physiology, evidenced by the capacity of imaged cells to enter and exit the S-phase, as well as undergo cell division.

Název v anglickém jazyce

What’s broken needs fixing: In-vivo analysis of DNA repair in plants using microirradiation and time-lapse imaging techniques

Popis výsledku anglicky

While we know that crucial elements of DNA repair pathways are evolutionary conserved from plants to humans, relatively little is known about the precise timing of the different DNA repair steps. DNA lesions need to be first recognized by dedicated protein complexes (e.g. the MRE11-RAD50-NBS1; MRN complex), the chromatin structure subsequently remodelled to allow access for repair enzymes, and finally the gaps need to be sealed and the chromatin structure restored. This whole processed can be mapped using biochemical studies and gene expression studies (e.g. gene upregulation after treatment with genotoxic agents). Laser microirradiation, first introduced in mammalian cell lines (cit, cit) is a technique that uses high power lasers to induce DNA damage in a defined cellular region using a confocal microscopy system, offering the possibility to study DNA repair in-vivo in unperturbed cells and has been routinely used in cell lines to study the function of different proteins in DNA repair. We have recently adapted this technique for use in plant systems (Nespor-Dadejova et al., 2022), which required several optimization steps that related to obstacles such as tissue thickness, light scattering and fragility of plant protoplasts. We have shown that the recruitment of factors such as the DNA clamp PCNA, and recognition factors such as MRE11 or PARP1 occurs in the matter of seconds after damage induction in plant systems, with dynamic recruitment to the sites of DNA lesions as evidenced by fluorescence recovery after photobleaching. In a new chapter of this research, we study DNA repair in the context of plant tissues (e.g. seedling roots), where we monitor locally damaged roots in a large temporal window, studying when and if the damaged cells are able to recover. Our first results indicate surprisingly dynamic behavior of the damaged sites. Importantly, we establish a system for root imaging that does not interfere with cellular physiology, evidenced by the capacity of imaged cells to enter and exit the S-phase, as well as undergo cell division.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10611 - Plant sciences, botany

Projekt

<a href="/cs/project/EH22_008%2F0004581" target="_blank" >EH22_008/0004581: Nové poznatky pro plodiny nové generace</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2025

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů