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

J 2022

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

NEŠPOR DADEJOVÁ, Martina, Michal FRANEK a Martina DVOŘÁČKOVÁ

Základní údaje

Originální název

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

Autoři

NEŠPOR DADEJOVÁ, Martina (203 Česká republika, domácí), Michal FRANEK (703 Slovensko, domácí) a Martina DVOŘÁČKOVÁ (203 Česká republika, garant, domácí)

Vydání

New Phytologist, Blackwell Science, 2022, 0028-646X

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10611 Plant sciences, botany

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 9.400

Kód RIV

RIV/00216224:14740/22:00127898

Organizační jednotka

Středoevropský technologický institut

DOI

http://dx.doi.org/10.1111/nph.18086

UT WoS

000785919300001

Klíčová slova anglicky

Arabidopsis; BiFC; DNA damage; DNA repair; microirradiation; PCNA; protoplasts

Štítky

CF CELLIM, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 8. 10. 2024 10:31, Ing. Martina Blahová

Anotace

V originále

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.

Návaznosti

EF16_026/0008446, projekt VaV

Název: Integrace signálu a epigenetické reprogramování pro produktivitu rostlin

GJ19-11880Y, projekt VaV

Název: Chromatinové prostředí indukované genotoxickými látkami

Investor: Grantová agentura ČR, Chromatinové prostředí indukované genotoxickými látkami

LTC18048, projekt VaV

Název: Prostorové uspořádání významných jaderných domén a jejich proteinových složek

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Prostorové uspořádání významných jaderných domén a jejich proteinových složek, INTER-COST

LTC20003, projekt VaV

Název: Dynamics of telomeres and rDNA loci in plant cell nuclei

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Dynamics of telomeres and rDNA loci in plant cell nuclei, INTER-COST

90129, velká výzkumná infrastruktura

Název: Czech-BioImaging II

Citovat

NEŠPOR DADEJOVÁ, Martina, Michal FRANEK a Martina DVOŘÁČKOVÁ. Laser microirradiation as a versatile system for probing protein recruitment and protein-protein interactions at DNA lesions in plants. New Phytologist. Blackwell Science, 2022, roč. 234, č. 5, s. 1891-1900. ISSN 0028-646X. Dostupné z: https://dx.doi.org/10.1111/nph.18086.

@article{2245593,
   author = {Nešpor Dadejová, Martina and Franek, Michal and Dvořáčková, Martina},
   article_number = {5},
   doi = {http://dx.doi.org/10.1111/nph.18086},
   keywords = {Arabidopsis; BiFC; DNA damage; DNA repair; microirradiation; PCNA; protoplasts},
   language = {eng},
   issn = {0028-646X},
   journal = {New Phytologist},
   title = {Laser microirradiation as a versatile system for probing protein recruitment and protein-protein interactions at DNA lesions in plants},
   url = {https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18086},
   volume = {234},
   year = {2022}
}

TY  - JOUR
ID  - 2245593
AU  - Nešpor Dadejová, Martina - Franek, Michal - Dvořáčková, Martina
PY  - 2022
TI  - Laser microirradiation as a versatile system for probing protein recruitment and protein-protein interactions at DNA lesions in plants
JF  - New Phytologist
VL  - 234
IS  - 5
SP  - 1891-1900
EP  - 1891-1900
PB  - Blackwell Science
SN  - 0028646X
KW  - Arabidopsis
KW  - BiFC
KW  - DNA damage
KW  - DNA repair
KW  - microirradiation
KW  - PCNA
KW  - protoplasts
UR  - https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18086
N2  - 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.
ER  -

NEŠPOR DADEJOVÁ, Martina, Michal FRANEK a Martina DVOŘÁČKOVÁ. Laser microirradiation as a versatile system for probing protein recruitment and protein-protein interactions at DNA lesions in plants. \textit{New Phytologist}. Blackwell Science, 2022, roč.~234, č.~5, s.~1891-1900. ISSN~0028-646X. Dostupné z: https://dx.doi.org/10.1111/nph.18086.

Podrobný výpis o publikaci