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. 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.

A Cancer Persistent Dna Repair Circuit Driven by Mdm2, Mdm4 (Mdmx), and Mutant P53 for Recruitment of Mdc1 and 53bp1 to Chromatin

“…However, 53BP1 also participates in reducing S-phase DNA damage and mitigating replication stress [59]. A maximal DNA damage response function requires MDC1-mediated recruitment of 53BP1 to chromatin through phospho-Ser139 H2AX [34]. However, when MDC1 interacts with chromatin in the absence of DNA damage, it does so in a ψH2AX-independent manner through a Proline-Serine-Threonine repeat (PST) domain that contributes to 53BP1 chromatin retention [34, 35].…”

“…We observed a dependence on mtp53 for the MDM2-53BP1 interaction. The interaction between 53BP1 and MDC1 is required for 53BP1 recruitment to stressed DNA in the absence of exogenous DNA damage through a Proline-Serine-Threonine repeat (PST) domain [34, 35], and we observed significantly reduced 53BP1-MDC1 foci in the absence of MDM2 protein, or when the MDM2-mtp53 interaction was inhibited using the small molecule inhibitor Nutlin 3a. These observations suggest that in the context of mtp53, the mtp53-MDM2-MDMX complex actively participates in regulating recruitment of 53BP1 to sites of DNA replication stress and endogenous DNA damage.…”

Shieldin complex assembly kinetics and DNA binding by SHLD3

“…Shieldin is believed to be a dynamic complex that might be hierarchically recruited to sites of DSBs 2,[6][7][8] . Central to the dynamic nature of the Shieldin complex, is the rare ability of REV7 to adopt multiple distinct native states.…”

“…The functions of SHLD1 and SHLD3 are unclear as they are biochemically uncharacterized. It has been proposed that Shieldin is not a constitutive complex, but rather that it is assembled and disassembled on-demand in cells [6][7][8] . Assembly of Shieldin might follow a linear hierarchy where SHLD3 localizes first to DNA breaks, followed by REV7 and SHLD2-SHLD1 in a 53BP1 and RIF1-dependent manner 2 .…”

Co-regulator activity of Mediator of DNA Damage Checkpoint 1 (MDC1) is associated with DNA repair dysfunction and PARP inhibitor sensitivity in lobular carcinoma of the breast

“…We profiled the MDC1 interactome in ILC cells to identify transcriptional co-regulator partners of ER:MDC1, and to assess differential MDC1 association with canonical DNA repair partners, by performing MDC1 co-immunoprecipitation + mass spectrometry (IP/MS) in ILC versus IDC cells. Of note, proteomic approaches have previously shown MDC1 association with the MRN complex and other DNA repair proteins, but studies to date have used exogenous and/or tagged versions of full-length MDC1 or MDC1 fragments, and have only been performed in HeLa, HEK293 (293), or U2OS cells (22)(23)(24)(25)(26). We performed endogenous MDC1 IP using a validated MDC1 antibody (see Methods) from ER+ cell lines MCF7 (IDC), HCC1428 (IDC, BRCA2-mutant), MM134 (ILC), and 44PE (ILC), as well as 293FT cells (to compare our approach to literature data).…”