Two distinct long-range synaptic complexes promote different aspects of end processing prior to repair of DNA breaks by non-homologous end joining

Abstract

Non-homologous end joining is the major double-strand break repair (DSBR) pathway in mammals. DNA-PK is the hub and organizer of multiple steps in non-homologous end joining (NHEJ). Recent high-resolution structures show how two distinct NHEJ complexes "synapse" two DNA ends. One complex includes a DNA-PK dimer mediated by XLF, whereas a distinct DNA-PK dimer forms via a domain-swap mechanism where the C terminus of Ku80 from one DNA-PK protomer interacts with another DNA-PK protomer in trans. Remarkably, the distance between the two synapsed DNA ends in both dimers is the same (∼115 Å), which matches the distance observed in the initial description of an NHEJ long-range synaptic complex. Here, a mutational strategy is used to demonstrate distinct cellular function(s) of the two dimers: one promoting fill-in end processing, while the other promotes DNA end resection. Thus, the specific DNA-PK dimer formed (which may be impacted by DNA end structure) dictates the mechanism by which ends will be made ligatable.

Keywords: DNA-PK; DNA-PKcs; DNA-dependent protein kinase; Ku; NHEJ; XLF; XRCC4; non-homologous end joining.

Figure 1. The C-terminal helix of Ku80 can interact with DNA-PKcs in both cis and trans, but ablating the cis interaction promotes end joining.

(A) Ribbon diagram of DNA-PK monomer and domain swap dimer. DNA-PKcs is shown in grey, Ku80 in green, Ku70 in orange and DNA in yellow. Diagram representing Ku80. A disordered linker (707-723) separates the C-terminal globular domain (595-706) from the C-terminal helix (724-732). (B) Amino acid sequence of the linker mutants. (C) The fluorescent substrate 290-Crimson/ZS was utilized to detect coding end joining of RAG-induced DSBs. Percent recombination of episomal fluorescent coding-end joining substrate in xrs6 cells transiently transfected with wild-type or mutant Ku80 expression constructs as indicated. Error bars indicate SEM from six independent experiments. ****, P<.0001; ***, P<.001 in two-way A NOVA with Holm-Sidak correction. Relative expression of each Ku80 mutant was assessed by immunoblotting. (D) + (E) xrs6 clonal transfectants expressing wild-type human Ku80 (WT), Δ6 linker shortening mutant (Δ6), insert 1 linker lengthening mutant (Ins1), Ctd linker/C-terminal+helix deletion mutant (ΔCtd), or empty vector (vect) were plated at cloning densities into complete medium with increasing doses of calicheamicin (D) or etoposide (E). Colonies were stained after eight days, and percent survival was calculated. Error bars represent the standard error of the means for six independent experiments.

Figure 2. Ablation of the two DNA-PK mediated dimers has distinct impacts on sensitivity to different radio-mimetic drugs.

(A + B) Ribbon diagrams of the domain-swap (PDB: 6ZHE) and XLF-mediated (PDB:7NFC) long-range complexes. DNA-PKcs is shown in grey, Ku80 in green, Ku70 in orange and DNA in yellow (left). Insert shows interaction of 4 basic residues with the Ku80 C-terminal helix. (C-H) Cartoon depicting end structures generated by drugs utilized. “x” denotes 3’PG adduct. Red line denotes peptide adduct on 4bp 5’ overhang. V3 clonal transfectants expressing wild-type or mutant DNA-PKcs as indicated were plated at cloning densities into complete medium with increasing doses of zeocin, calicheamicin, teniposide, etoposide, or camptothecin (in the presence of 20 μM KU55933). Colonies were stained after eight days, and percent survival was calculated. Error bars represent the standard error of the mean for at least three independent experiments. (G) Cartoon depicting toxic NHEJ. (I) Immunoblot for DNA-PKcs expression in V3 transfectants.

Figure 3. Cells expressing the domain-swap mutant are deficient in the TDP2-independent, nuclease dependent pathway for processing peptide-blocked 5’overhangs.

(A) Diagram of repair of TOP2 poison-induced DNA damage. (B) Diagram of CRISPR strategy to ablate TDP2 from V3 transfectants. (right) Agarose gel electrophoresis of PCR to detect genomic deletions within in V3 cells. (bottom) Immunoblot for DNA-PKcs expression in V3 transfectants before and after TDP2 ablation. (C) V3 clonal transfectants expressing wild-type or mutant DNA-PKcs as indicated were plated at cloning densities into complete medium with increasing doses of calicheamicin or teniposide. Colonies were stained after eight days, and percent survival was calculated. Error bars represent the standard error of the means for six independent experiments.

Figure 4. Disruption of the two long-range synaptic complexes have opposite effects on DNA end-processing.

(A) Cartoon depicting the alt-VDJ substrate; restoration of the GFP reading frame requires nucleotide deletions of 10bp from either coding end and utilization of 9bp of short sequence homology. The fluorescent substrate 290-Crimson/ZS (coding joints) or alt-VDJ were utilized to detect coding end joining or alt-VDJ joining of hyper RAG mutant-induced DSBs in V3 cells expressing wild-type, 4Xala mutant, or 898/2659 mutant DNA-PKcs. With the alt-VDJ substrate, dsRED expression was co-transfected to control for transfection efficiency. Percent recombination represents %GFP/%RFP. (B) Cartoon depicting the Fill-in substrate that when cleaved with appropriate restriction enzymes will only restore Crimson expression if over-hanged ends are filled in and re-ligated to blunt end. Uncut or cleaved Fill-in substrate was transfected into cells expressing wild-type DNA-PKcs, the 4Xala, or 898/2569 mutants. Crimson and GFP expression was assessed by Flow cytometry 72 hours later and Crimson/GFP is expressed as %perfect joining. For (A+B) error bars indicate SEM from three independent experiments. **P<0.01; ****P<0.0001; ns=not significant in two-way ANOVA with Holm-Sidak correction. (C) 2.5% agarose electrophoresis of PCR amplification of coding joints from AT coding joint substrate from V3 stable transfectants expressing wild-type or mutant DNA-PKcs and then transfected with substrate and RAG1 and RAG2 expression constructs. Cells were harvested 72 hours after transfection; coding joint substrate was isolated by alkaline lysates, followed by PCR for coding joints. (D+E) Summary of amplicon sequencing of coding joints amplified from V3 transfectants expressing wild type, 4xAla, or 898/2569 DNA-PKcs. Results are averages of two separate experiments.

Figure 5. Cells expressing mutants that disrupt both long-range synaptic complexes are similarly resistant to DNA damaging agents as cells expressing wild-type DNA-PKcs.

V3 clonal transfectants expressing wild-type human DNA-PKcs, no DNA-PKcs, or mutant DNA-PKcs as indicated were plated at cloning densities into complete medium with increasing doses of calicheamicin (A), etoposide (B), or camptothecin (in the presence of 20 μM KU55933) (C). Colonies were stained after eight days, and percent survival was calculated. Error bars represent the standard error of the means for six independent experiments. (D) Immunoblot for DNA-PKcs expression in V3 transfectants. (E) 2.5% agarose electrophoresis of PCR amplification of coding joints from AT coding joint substrate from V3 stable transfectants expressing or not wild-type or mutant DNA-PKcs and then transfected with substrate and RAG1 and RAG2 expression constructs.

Figure 6. More disruptive mutants designed to disrupt the domain-swap and XLF-mediated DNA-PK dimers enhance their cellular phenotypes.

(A) (left) The fluorescent substrate 290-Crimson/ZS (coding joints) was utilized to detect coding end joining of RAG-induced DSBs in V3 cells expressing wild-type or mutant DNA-PKcs as depicted. Percent recombination represents %GFP/%Crimson. **P<0.01; ****P<0.0001; ns=not significant in two-way ANOVA with Holm-Sidak correction, (right) Immunoblot depicting relative DNA-PKcs expression in stable V3 clones as indicated. (B+C) V3 clonal transfectants expressing wild-type DNA-PKcs, vector control or DNA-PKcs mutants as indicated were plated at cloning densities into complete medium with increasing doses of calicheamicin (B) or teniposide (C). Colonies were stained after eight days, and percent survival was calculated. Error bars represent the standard error of the means for six independent experiments. (C) Immunoblot analyses using indicated antibodies of cell extracts from indicated V3 transfectants treated or not with 40nM calicheamicin and 1uM Okadaic acid for thirty minutes. * Indicates a non-specific band.

Figure 7

Iterative model of NHEJ.