Repair of DNA double strand breaks (DSBs) is integral to preserving genomic integrity. Therefore, defining the mechanisms underlying DSB repair will enhance our understanding of how defects in these pathways contribute to human disease and could lead to the discovery of new approaches for therapeutic intervention. Here, we established a panel of HaloTagged DNA damage response factors in U2OS cells which enables concentration-dependent protein labeling. Genomic insertion of the HaloTag at the endogenous loci of the repair factors preserves expression levels and proteins retain proper subcellular localization, foci-forming ability, and functionally support DSB repair. We systematically analyzed total cellular protein abundance, measured recruitment kinetics to DSBs, and defined the diffusion dynamics and chromatin binding by live-cell single-molecule imaging. Our work demonstrates that the Shieldin complex, a critical factor in end joining, does not exist in a preassembled state and Shieldin components are recruited to DSBs with different kinetics. Additionally, live-cell single-molecule imaging revealed the constitutive interaction between MDC1 and chromatin mediated by the PST repeat domain of MDC1. Altogether, our studies demonstrate the utility of single-molecule imaging to provide mechanistic insights into DNA repair, which will serve as a powerful resource for characterizing the biophysical properties of DNA repair factors in living cells.

Repair of DNA double strand breaks (DSBs) is integral to preserving genomic integrity. Therefore, defining the mechanisms underlying DSB repair will enhance our understanding of how defects in these pathways contribute to human disease and could lead to the discovery of new approaches for therapeutic intervention. Here, we established a panel of HaloTagged DNA damage response factors in U2OS cells which enables concentration-dependent protein labeling by fluorescent HaloTag ligands. Genomic insertion of HaloTag at the endogenous loci of these repair factors preserves expression levels and proteins retain proper subcellular localization, foci-forming ability, and functionally support DSB repair. We systematically analyzed total cellular protein abundance, measured recruitment kinetics to laser-induced DNA damage sites, and defined the diffusion dynamics and chromatin binding characteristics by live-cell single-molecule imaging. Our work demonstrates that the Shieldin complex, a critical factor in end-joining, does not exist in a preassembled state and that relative accumulation of these factors at DSBs occurs with different kinetics. Additionally, live-cell single-molecule imaging revealed the constitutive interaction between MDC1 and chromatin mediated by its PST repeat domain. Altogether, our studies demonstrate the utility of single-molecule imaging to provide mechanistic insights into DNA repair, which will serve as a powerful resource for characterizing the biophysical properties of DNA repair factors in living cells.

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Non-homologous end joining (NHEJ) is the predominant path-way that repairs DNA double-stranded breaks (DSBs) in vertebrates. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed approximately 115Å apart in one of two long-range synaptic complexes. The first long-range complex can be formed with only the KU70/80 heterodimer and DNA-PKcs while the second long-range complex also includes XRCC4, XLF, and Ligase 4. Both long-range complexes inefficiently progress to short-range synaptic complexes that juxtapose DNA ends to facilitate ligation. Here we perform singlemolecule analyses of the recruitment of Halo-tagged NHEJ factors to DSBs. Our results provide direct evidence for stepwise maturation of NHEJ complex and precisely define kinetics of core NHEJ factor binding to DSBs in living cells.

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