New Methodologies to Study DNA Repair Processes in Space and Time Within Living Cells

Abstract

DNA repair requires a coordinated effort from an array of factors that play different roles in the DNA damage response from recognizing and signaling the presence of a break, creating a repair competent environment, and physically repairing the lesion. Due to the rapid nature of many of these events, live-cell microscopy has become an invaluable method to study this process. In this review we outline commonly used tools to induce DNA damage under the microscope and discuss spatio-temporal analysis tools that can bring added information regarding protein dynamics at sites of damage. In particular, we show how to go beyond the classical analysis of protein recruitment curves to be able to assess the dynamic association of the repair factors with the DNA lesions as well as the target-search strategies used to efficiently find these lesions. Finally, we discuss how the use of mathematical models, combined with experimental evidence, can be used to better interpret the complex dynamics of repair proteins at DNA lesions.

Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

“…Ensemble laser microirradiation is one of the most widely used methods for generating localized DNA breaks to study the biological response to DNA damage in live cells. 53 , 75 We and others have obtained valuable insights into the recruitment of PARP1/2, demonstrating that endogenous and overexpressed PARP1 accumulates significantly faster than PARP2 at laser-induced DNA lesions ( Figure S1 E, Table S1 ). 2 , 3 , 14 , 76 More recent work using ensemble microirradiation combined with FRAP suggests that PARP1 undergoes rapid turnover at sites of DNA lesions, 42 challenging the concept of PARP trapping.…”

Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

“…Bulk laser microirradiation is one of the most widely used methods for generating localized DNA breaks to study the biological response to DNA damage in live cells (Mahadevan et al, 2019a; Zentout et al, 2021). We and others have obtained valuable insights into the recruitment of PARP1/2, demonstrating that endogenous and overexpressed PARP1 accumulates significantly faster than PARP2 at laser induced DNA lesions (Figure S1E, Table S1) (Caron et al, 2019; Haince et al, 2008; Mahadevan et al, 2019b; Mortusewicz et al, 2007).…”

Genome editing in macroalgae: advances and challenges

“…Notably, U. prolifera exhibited a high incidence of substitution mutations, which is extremely rare in plants or animals ( Hwang et al, 2020 ; Zhang et al, 2020 ). A lot of work on DNA repair has been done in other organisms, with many published protocols that can be leveraged here as well ( Bjergbæk, 2016 ; Zentout et al, 2021 ; van de Kooij and van Attikum, 2022 ). In mammalian systems the successful redirection of DNA repair pathways has already been demonstrated with chemical inhibitors ( Maruyama et al, 2015 ), cell cycle synchronization ( Lin et al, 2014 ), Homology-directed repair (HDR) template modifications ( Cruz-Becerra and Kadonaga, 2020 ; Schubert et al, 2021 ), modulation of regulatory factors ( Canny et al, 2018 ; Charpentier et al, 2018 ; Jayavaradhan et al, 2019 ), and engineered Cas9 variants ( Chauhan et al, 2023 ).…”