Systematic analysis of the molecular and biophysical properties of key DNA damage response factors

Abstract

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.

Diffusion of activated ATM explains γH2AX and MDC1 spread beyond the DNA damage site

“…In this line, the tagged MDC1 is expressed at near-physiological levels 16 under cell-cycle control. Our results (Figures 1A and 1B; Movie S1) show that MDC1 is quickly recruited to the site of micro-IR 1,17 , with a half-time of 55s, reaching its maximum levels at around 900s, whereafter it spreads around the complex lesion. As a result, MDC1 is heavily depleted from other regions of the nucleus following damage induction.…”

“…MDC1 mobility was approximately an order of magnitude slower (average D=0.055±0.033μm 2 /s) than expected for the pure diffusion of a 200kDa (0.4-0.6μm 2 /s) protein. It is known that MDC1 is weakly bound to the chromatin even in the absence of DNA damage 17,18 . This can also be inferred from the irregular spatial distribution of MDC1, which resembles the spatial structure of chromatin.…”

Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells

“…As previously described, the expression level of Ku70 is very high (1.3 -1.6x10 6 molecules per cell) 20 .The expression levels of XLF (54 -61x10 3 molecules per cell) and XRCC4 (23 -28x10 3 molecules per cell) were substantially lower. In previous work we determined that DNA-PKcs is present at 120x10 6 molecules per cell 17 . Together these observations demonstrate that the abundance of the core NHEJ factors spans two orders of magnitude, and the components of the initial end-binding complex (Ku70 and DNA-PKcs) are present in at least 10-fold excess over the components of the ligation competent short-range complex (XLF and XRCC4), taking into account that XRCC4 acts as a dimer and XLF is largely excluded from the nucleus.…”

“…NHEJ factors. To facilitate live cell single-molecule imag-ing of NHEJ factors, CRISPR-Cas9 and homology-directed repair were used to insert the coding sequence for a 3xFLAG-HaloTag (Halo) at the N-terminus of the endogenous loci encoding Ku70, DNA-PKcs (generated in our previous work 17 ), XLF, and XRCC4 in U2OS cells (Fig. 1A).…”

“…3B) 23 . As controls for freely diffusing and chromatin bound proteins, constructs encoding only the HaloTag fused to three nuclear localization sequences (Halo-NLS) and HaloTag-histone H2B (Halo-H2B) were transfected into parental U2OS cells 17 .…”