Laser Microirradiation and Real-time Recruitment Assays Using an Engineered Biosensor

Background

DNA double-strand breaks (DSBs) are a threat to genome integrity and stability. DSBs can be generated in response to external agents (e.g., irradiation, radiomimetic chemicals) or formed as byproducts of normal cell metabolism (e.g., oxidative stress, DNA replication errors). To deal with DSBs, eukaryotic cells use complex repair machinery equipped with protein effectors responsible for detecting, signaling, and promoting the repair of these lesions. Defects in DNA DSB repair can lead to mutations or chromosomal aberrations and, ultimately, contribute to the onset of cancer and neurodegenerative diseases (Jackson and Bartek, 2009; Ciccia and Elledge, 2010; Polo and Jackson, 2011).

In response to DNA DSBs, the serine/threonine kinase ataxia-telangiectasia mutated (ATM) is activated and promotes the phosphorylation of various proteins in chromatin surrounding the damage sites. These phosphorylation substrates include histone variant H2AX in vertebrates. Phosphorylated H2AX (γH2AX) leads to the sequential recruitment of mediator of DNA damage checkpoint protein 1 (MDC1) and the E3 ubiquitin ligases RNF8 and RNF168. RNF168 specifically ubiquitinates lysines 13 or 15 at the N-terminal tail of histone H2A (H2AK13/15Ub) (Mattiroli et al., 2012), which in turn can recruit effectors of non-homologous end joining (NHEJ) or homologous recombination (HR)-mediated repair (Mattiroli and Penengo, 2021). The NHEJ repair factor 53BP1 is recruited by nucleosomes containing both H4K20me2 and H2AK15Ub (Fradet-Turcotte et al., 2013; Wilson et al., 2016; Hu et al., 2017). In contrast, the BRCA1/BARD1 complex promotes the HR pathway and is recruited by nucleosomes modified with H2AK13Ub or H2AK15Ub, only if H4K20 is not methylated (H4K20Me0) (Becker et al., 2021; Hu et al., 2021). The minimal focus-forming region (FFR) of 53BP1 has been previously shown to localize to DSBs induced by gamma or laser irradiation, and thereby report local increases in H2AK15Ub at DNA lesions (Fradet-Turcotte et al., 2013; dos Santos Passos et al., 2021). However, structural and biochemical studies have demonstrated that 53BP1 binding to the nucleosomes requires both H2AK15Ub and H4K20Me2 modifications. The H4K20Me2 modifications are enriched in the G1 and early S-phases of the cell cycle (Fradet-Turcotte et al., 2013; Wilson et al., 2016; Hu et al., 2017; Michelena et al., 2021).

In recent work, we developed and validated an avidity-based sensor to detect H2AK13/15Ub-nucleosomes. This sensor, which we call Reader1.0, can bind nucleosomes containing either H2AK13Ub or H2AK15Ub in vitro, and it localizes to sites of RNF168-mediated ubiquitination in mammalian cells. In addition, Reader1.0 binding to H2AK13/15Ub-nucleosomes does not depend on the methylation status of histone H4K20; i.e., Reader1.0 recognizes H2AK13/15Ub-nucleosomes containing either H4K20me0 or H4K20me2. When fused to fluorescent proteins and expressed at tightly-controlled levels, Reader1.0 can be used to monitor the dynamics of H2AK13/15Ub in live cells over long time intervals without causing significant perturbation of cell growth rate or DNA repair kinetics (dos Santos Passos et al., 2021).

The protocol presented here describes how to measure the recruitment of readers of RNF168-dependent H2AK13/15 ubiquitination to DNA DSBs. To rapidly generate local high concentrations of H2AK13/15Ub, laser microirradiation was used to induce DNA damage in the nuclei of U-2 OS cells. The main advantages of this method are the abilities to target defined nuclear volumes and to generate similar amounts of DNA damage in different nuclei within a cell population (Bekker-Jensen et al., 2006; Lukas et al., 2003). By co-expressing mCherry-53BP1-FFR and Reader1.0-EGFP, we demonstrated that our designed sensor Reader1.0 and 53BP1-FFR both redistribute to laser-induced DNA lesions with similar kinetics. In sum, this protocol describes the detection of H2AK13/15Ub at DNA DSBs in live cells by fluorescence confocal microscopy.