Abstract
To ward off against the catastrophic consequences of persistent DNA double-strand breaks (DSBs), eukaryotic cells have developed a set of complex signaling networks that detect these DNA lesions, orchestrate cell cycle checkpoints and ultimately lead to their repair. Collectively, these signaling networks comprise the DNA damage response (DDR). The current knowledge of the molecular determinants and mechanistic details of the DDR owes greatly to the continuous development of ground-breaking experimental tools that couple the controlled induction of DSBs at distinct genomic positions with assays and reporters to investigate DNA repair pathways, their impact on other DNA-templated processes and the specific contribution of the chromatin environment. In this review, we present these tools, discuss their pros and cons and illustrate their contribution to our current understanding of the DDR.
Highlights
Specialty section: This article was submitted to Cellular Biochemistry, a section of the journal Frontiers in Molecular Biosciences
The current knowledge of the molecular determinants and mechanistic details of the DNA damage response (DDR) owes greatly to the continuous development of ground-breaking experimental tools that couple the controlled induction of double-strand breaks (DSBs) at distinct genomic positions with assays and reporters to investigate DNA repair pathways, their impact on other DNAtemplated processes and the specific contribution of the chromatin environment. We present these tools, discuss their pros and cons and illustrate their contribution to our current understanding of the DDR
The DDR requires the activation of the ATM kinase, a member of the phosphoinositide 3-kinase (PI3K)-related protein kinase family (Blackford and Jackson, 2017), which is rapidly recruited to chromatin in response to DSBs through the interaction with the MRE11-RAD50-NBS1 (MRN) complex
Summary
In order to bypass the need for introducing a transgene and to avoid potential, non-generalizable, side effects of transgenic loci on the repair process (e.g., in the case of LacI repeats, a high copy number triggering a peculiar chromatin state), efforts have been made recently to develop alternative systems where DSBs can be induced at endogenous, annotated loci on the genome (Figure 4). The Legube lab, fused AsiSI to a modified ER ligand-binding domain, which controls nuclear localization of AsiSI–ER fusion protein, and to an auxin-inducible degron enabling controlled ubiquitination and degradation of the enzyme (Iacovoni et al, 2010; Massip et al, 2010; Aymard et al, 2014) Stable integration of this construct in the genome of U2OS cells generated a DSB inducible via AsiSI (DIvA) system, where multiple annotated DSBs can be induced after 4-OHT treatment and DNA repair accurately monitored following auxin treatment. CRISPR/Cas The discovery of the CRISPR/Cas system in 2013 strongly revolutionized the DDR field by providing the ability to introduce DSBs at annotated loci, in a simple and efficient manner, by the mean of a small guide RNA embedded in the Cas nuclease This approach has been used successfully to induce DSBs and study DNA repair in rDNA (van Sluis and McStay, 2015; Korsholm et al, 2019). A major challenge is to refine these DSBinducible systems and the subsequent methodologies to analyze repair in order to overcome these limitations
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