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

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Publikace nespadá pod Ekonomicko-správní fakultu, ale pod Středoevropský technologický institut. Oficiální stránka publikace je na webu muni.cz.
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NEŠPOR DADEJOVÁ Martina FRANEK Michal DVOŘÁČKOVÁ Martina

Rok publikování 2022
Druh Článek v odborném periodiku
Časopis / Zdroj New Phytologist
Fakulta / Pracoviště MU

Středoevropský technologický institut

Citace
www https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18086
Doi http://dx.doi.org/10.1111/nph.18086
Klíčová slova Arabidopsis; BiFC; DNA damage; DNA repair; microirradiation; PCNA; protoplasts
Popis 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.
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