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Doctoral thesis - Peer-reviewed, 2022

Characterization of chromatin mobility upon DNA damage in Arabidopsis thaliana

Meschichi, Anis

Abstract

Plant cells are subject to high levels of DNA damage from dependence on sunlight for energy and the associated exposure to biotic and abiotic stresses. Double-strand breaks (DSBs) are a particularly deleterious type of DNA damage, potentially leading to chromosome rearrangements or loss of entire chromosome arms. The presence of efficient and accurate repair mechanisms may be particularly important for sedentary organisms with late separation of the germline, such as plants. DSB repair is accomplished by two main pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ is achieved by stabilization and re-ligation of broken DNA ends, often with a loss or mutation of bases. HR is a more complex and conservative mechanism in which intact homologous regions are used as a template for repair. The molecular mechanisms that control DSB signaling and repair have been characterized extensively. Nonetheless, little is known about how the homology search happens in the crowded space of the cell nucleus. This thesis reveals the methodology to capture chromatin motion to investigate nuclear dynamics in different developmental and cellular contexts. Using live imaging approaches, we measured chromosome mobility by tracking the motion of specific loci using the lacO/LacI and ParB/parS tagging systems in Arabidopsis thaliana.


Our results have shown that chromatin mobility is affected by cell differentiation level, cell cycle phase, or genomic position, and that chromatin mobility increases when DNA damage is induced. Moreover, we observed an increase in chromatin mobility upon the induction of DNA damage, specifically at the S/G2 phases of the cell cycle. Importantly, this increase in mobility in S/G2 was lost on sog1-1 mutant, a central transcription factor of the DNA damage response (DDR), indicating that repair mechanisms actively regulate chromatin mobility upon DNA damage. Studies have shown that HR is the predominant DSB repair pathway occurring during S/G2 phase. Therefore, we investigated the mobility of two GFP-tagged HR regulators, RAD51 and RAD54, corresponding to early and late HR. DSB sites show remarkably high mobility levels at the early HR stage. Subsequently, a drastic decrease in DSB mobility is observed, which seems to be associated with the relocation of DSBs to the nucleus periphery.


Altogether, our study suggests chromatin mobility as a non-negligible factor for DNA repair in plants, which may facilitate physical searching in the nuclear space thereby helping to locate a homologous template during homology-directed DNA repair.

Keywords

Arabidopsis; chromatin mobility; DNA damage; SOG1; RAD51; homologous recombination; cell cycle; chromatin dynamic; mean square displacement; DNA damage response

Published in

Acta Universitatis Agriculturae Sueciae
2022, number: 2022:50
ISBN: 978-91-7760-975-9, eISBN: 978-91-7760-976-6
Publisher: Swedish University of Agricultural Sciences

Authors' information

Swedish University of Agricultural Sciences, Department of Plant Biology

UKÄ Subject classification

Agricultural Science

URI (permanent link to this page)

https://res.slu.se/id/publ/118378