Laser microirradiation as a versatile system for probing protein recruitment and protein-protein interactions at DNA lesions in plants

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14740%2F22%3A00127898" target="_blank" >RIV/00216224:14740/22:00127898 - isvavai.cz</a>

Výsledek na webu

<a href="https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18086" target="_blank" >https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18086</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1111/nph.18086" target="_blank" >10.1111/nph.18086</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Laser microirradiation as a versatile system for probing protein recruitment and protein-protein interactions at DNA lesions in plants

Popis výsledku v původním jazyce

Plant protoplasts are generated by treatment with digestion enzymes, producing plant cells devoid of the cell wall and competent for efficient polyethylene glycol mediated transformation. This way fluorescently tagged proteins can be introduced to the protoplasts creating an excellent system to probe the localization and function of uncharacterized plant proteins in vivo. We implement the method of laser microirradiation to generate DNA lesions in Arabidopsis thaliana, which enables monitoring the recruitment and dynamics of the DNA repair factors as well as bimolecular fluorescence complementation assay to test transient, conditional interactions of proteins directly at sites of DNA damage. We demonstrate that laser microirradiation in protoplasts yields a physiological cellular response to DNA lesions, based on proliferating cell nuclear antigen (PCNA) redistribution in the nucleus and show that factors involved in DNA repair, such as MRE11 or PCNA are recruited to induced DNA lesions. This technique is relatively easy to adopt by other laboratories and extends the current toolkit of methods aimed to understand the details of DNA damage response in plants. The presented method is fast, flexible and facilitates work with different mutant backgrounds or even different species, extending the utility of the system.

Název v anglickém jazyce

Laser microirradiation as a versatile system for probing protein recruitment and protein-protein interactions at DNA lesions in plants

Popis výsledku anglicky

Plant protoplasts are generated by treatment with digestion enzymes, producing plant cells devoid of the cell wall and competent for efficient polyethylene glycol mediated transformation. This way fluorescently tagged proteins can be introduced to the protoplasts creating an excellent system to probe the localization and function of uncharacterized plant proteins in vivo. We implement the method of laser microirradiation to generate DNA lesions in Arabidopsis thaliana, which enables monitoring the recruitment and dynamics of the DNA repair factors as well as bimolecular fluorescence complementation assay to test transient, conditional interactions of proteins directly at sites of DNA damage. We demonstrate that laser microirradiation in protoplasts yields a physiological cellular response to DNA lesions, based on proliferating cell nuclear antigen (PCNA) redistribution in the nucleus and show that factors involved in DNA repair, such as MRE11 or PCNA are recruited to induced DNA lesions. This technique is relatively easy to adopt by other laboratories and extends the current toolkit of methods aimed to understand the details of DNA damage response in plants. The presented method is fast, flexible and facilitates work with different mutant backgrounds or even different species, extending the utility of the system.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10611 - Plant sciences, botany

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

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

Rok uplatnění

2022

Kód důvěrnosti údajů

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

Název periodika

New Phytologist

ISSN

0028-646X

e-ISSN

—

Svazek periodika

234

Číslo periodika v rámci svazku

5

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

10

Strana od-do

1891-1900

Kód UT WoS článku

000785919300001

EID výsledku v databázi Scopus

2-s2.0-85129048753