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 and Martina DVOŘÁČKOVÁ

Basic information

Original name

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

Authors

NEŠPOR DADEJOVÁ, Martina (203 Czech Republic, belonging to the institution), Michal FRANEK (703 Slovakia, belonging to the institution) and Martina DVOŘÁČKOVÁ (203 Czech Republic, guarantor, belonging to the institution)

Edition

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

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10611 Plant sciences, botany

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 9.400

RIV identification code

RIV/00216224:14740/22:00127898

Organization unit

Central European Institute of Technology

DOI

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

UT WoS

000785919300001

Keywords in English

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

Abstract

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.

Links

EF16_026/0008446, research and development project

Name: Integrace signálu a epigenetické reprogramování pro produktivitu rostlin

GJ19-11880Y, research and development project

Name: Chromatinové prostředí indukované genotoxickými látkami

Investor: Czech Science Foundation, Chromatin environment induced by genotoxic agents

LTC18048, research and development project

Name: Prostorové uspořádání významných jaderných domén a jejich proteinových složek

Investor: Ministry of Education, Youth and Sports of the CR, Spatial organisation of key nuclear domains and their protein components, INTER-COST

LTC20003, research and development project

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

Investor: Ministry of Education, Youth and Sports of the CR, INTER-COST

90129, large research infrastructures

Name: Czech-BioImaging II

Citovat

NEŠPOR DADEJOVÁ, Martina, Michal FRANEK and 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, vol. 234, No 5, p. 1891-1900. ISSN 0028-646X. Available from: 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 and 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, vol.~234, No~5, p.~1891-1900. ISSN~0028-646X. Available from: https://dx.doi.org/10.1111/nph.18086.

Detailed Information on Publication Record