Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency

Abstract

The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PK_cs and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5' overhangs, and 3' overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed.

Keywords: DNA damage; DNA recombination; DNA repair; chromosomes; double-strand DNA break; enzyme; ligase; nuclease.

FIGURE 1.

NHEJ reconstitution workflow. NHEJ proteins are added to labeled DNA substrate, and NHEJ products are resolved by denaturing PAGE. NHEJ products are cut out and PCR-amplified. PCR products are TA cloned into a pGEM-T vector and transformed into E. coli. Vector is then isolated from colonies, and the junctions are sequenced.

FIGURE 2.

NHEJ of resection-dependent compatible 3′ overhang requires Artemis and is strongly stimulated by Ku and DNA-PK_cs. NHEJ proteins (50 nm Ku, 25 nm DNA-PK_cs, 25 nm Artemis, 100 nm X4·LIV) were incubated for 60 min at 37 °C with 20 nm *HC101/102 and 20 nm pHC115/116 in a reaction buffer containing 200 nm streptavidin to bind to biotin (B) to block one end of the DNA. In addition, p represents a 5′ phosphate, and the asterisk represents the radiolabel. NHEJ efficiencies are noted underneath. The reported values are averages of three independent experiments with a S.E. of 0.17% (lane 5), 1.0% (lane 7), 2.1% (lane 8), and 4.4% (lane 9).

FIGURE 3.

Artemis resection of 3′ overhangs is stimulated by X4·LIV. NHEJ proteins were incubated for 60 min at 37 °C with a reaction buffer containing 200 nm streptavidin to bind to biotin (B) to block one end of the DNA. In addition, p represents a 5′ phosphate, and the asterisk represents the radiolabel. A, 20 nm *HC101/102 was incubated with 50 nm Artemis (lane 2); 25 nm DNA-PK_cs and 50 nm Artemis (lane 3); 50 nm Artemis and 100 nm X4·LIV (lane 4); and 25 nm DNA-PK_cs, 50 nm Artemis, and 100 nm X4·LIV (lane 5). B, *20 nm HC 101/102 was incubated with 20 nm pHC115/116 and 50 nm Ku, 25 nm DNA-PK_cs, 25 nm Artemis, and 100 nm X4·LIV as indicated.

FIGURE 4.

NHEJ of resection-dependent compatible 3′ overhangs is strongly dependent on microhomology. NHEJ proteins (50 nm Ku, 25 nm DNA-PK_cs, 25 nm Artemis, 100 nm X4·LIV) were incubated with 20 nm *HC101/102 and either 20 nm pHC115/116 (lanes 1- 6), pHC115/123 (lanes 7- 11), pHC115/124 (lanes 12- 16), or pHC115/120 (lanes 17- 21) for 60 min at 37 °C in a reaction containing 200 nm streptavidin to bind to biotin (B) to block one end of the DNA. In addition, p represents a 5′ phosphate, and the asterisk represents the radiolabel. NHEJ efficiencies are noted underneath. The S.E. values from independent experiments are 0.05% (lane 3), 0.55% (lane 4), 1.75% (lane 5), 0.4% (lane 6), 0.05% (lane 8), 0.1% (lane 9), 0.35% (lane 10), 1.45% (lane 11), 0.1% (lane 13), 0.1% (lane 14), 0.05% (lane 15), 0.15% (lane 16), 0.05% (lane 18), 0.05% (lane 19), 0.05% (lane 20), and 0.05% (lane 21). Variations in total recovery are less marked in replicates of the same experiment, and the conversion of substrate to product is unaffected by variations in total recovery.

FIGURE 5.

The Ku, DNA-PK_cs, Artemis, and X4·LIV complex is not sufficient for NHEJ of incompatible DNA ends, whereas blunt-ended DNA only requires Ku and X4·LIV. NHEJ proteins (50 nm Ku, 25 nm DNA-PK_cs, 25 nm Artemis, 100 nm X4·LIV) were incubated for 60 min at 37 °C in a reaction buffer containing 200 nm streptavidin to bind to biotin (B) to block one end of the DNA. In addition, p represents a 5′ phosphate, and the asterisk represents the radiolabel. NHEJ efficiencies are noted underneath. A, DNA substrates used were 20 nm *HC101/102 and 20 nm pHC115/119. B, DNA substrates used were 20 nm *HC101/102 and 20 nm pHC115/120. C, DNA substrates used were 20 nm *HC121/102 and 20 nm pHC115/120. These are representative gels of three similar experiments that have confirmed these results.

FIGURE 6.

NHEJ of 3′ incompatible ends is stimulated by Pol μ but not Pol λ. NHEJ proteins (50 nm Ku, 25 nm DNA-PK_cs, 25 nm Artemis, 100 nm X4·LIV, 25 nm Pol μ, and 25 nm Pol λ) were incubated for 60 min at 37 °C in a reaction containing 200 nm streptavidin to bind to biotin (B) to block one end of the DNA. In addition, p represents a 5′ phosphate, and the asterisk represents the radiolabel. NHEJ efficiencies are noted underneath. A, DNA substrates used were 20 nm *HC101/102 and either 20 nm pHC115/116 (lanes 1–4), pHC115/119 (lanes 5–8), or pHC115/120 (lanes 9–12). Blunt end ligations were performed with 20 nm *HC121/102 and 20 nm pHC115/120 (lanes 13–16). B, 100 μm dNTPs were incubated with DNA substrates 20 nm *HC101/102 and either 20 nm pHC115/116 (lanes 1–4), pHC115/119 (lanes 5–8), or pHC115/120 (lanes 9–12). These are representative gels of multiple similar experiments that have confirmed these results.

FIGURE 7.

PAXX only stimulates blunt end NHEJ. NHEJ proteins (50 nm Ku, 25 nm DNA-PK_cs, 25 nm Artemis, 100 nm X4·LIV, 500 nm PAXX, 200 nm XLF, and 25 nm Pol μ) were incubated for 60 min at 37 °C in a reaction containing 200 nm streptavidin to bind to biotin (B) to block one end of the DNA. In addition, p represents a 5′ phosphate, and the asterisk represents the radiolabel. A, RD-compatible NHEJ was performed with 20 nm *HC101/102 and 20 nm pHC115/116. NHEJ efficiencies are noted underneath. The reported values are averages of three independent experiments with a S.E. of 0.1% (lane 2), 0.2% (lane 3), 0.6% (lane 4), and 0.4% (lane 5). B, DNA substrates used were 20 nm *HC101/102 and either 20 nm pHC115/119 (lanes 1–5) and pHC115/120 (lanes 6–10). Blunt end ligations were performed with 20 nm *HC121/102 and 20 nm pHC115/120 (lanes 11–15). NHEJ efficiencies are noted underneath. The reported values are averages of three independent experiments with a S.E. of 0.6% (lane 2), 0.3% (lane 3), 0.4% (lane 4), 0.6% (lane 5), 0.3% (lane 7), 0.3% (lane 8), 0.4% (lane 9), 0.2% (lane 10), 0.3% (lane 12), 0.4% (lane 13), 0.6% (lane 14), and 0.3% (lane 15).

FIGURE 8.

NHEJ of 5′ overhang to a blunt end is stimulated by XLF and PAXX with no polymerase effect. NHEJ proteins included 50 nm Ku, 25 nm DNA-PK_cs, 25 nm Artemis, 100 nm X4·LIV, 500 nm PAXX, 200 nm XLF. A, depiction of DNA substrate where p represents a 5′ phosphate, and the asterisk represents the radiolabel. Pol μ and Pol λ were incubated with 20 nm pJG277*/JG226-ddG for 60 min at 37 °C. NHEJ efficiencies are noted underneath. (Head) refers to the end with the 5′ overhang, and (Tail) refers to the blunt end. B, XLF and PAXX were varied in NHEJ reactions with Ku and X4·LIV (lanes 2–5); Ku, Artemis, and X4·LIV (lanes 6–9); and Ku, DNA-PK_cs, Artemis, and X4·LIV (lanes 10–13). C, Pol μ and Pol λ (25 nm each) were included in NHEJ reactions with Ku, DNA-PK_cs, Artemis, X4·LIV, XLF, and PAXX as noted. These are representative gels of multiple similar experiments that have confirmed these results.

FIGURE 9.

Diagram of end complex. This diagram shows how the various NHEJ proteins might associate at the ends. For simplification, we have only depicted the ligation of the top strand, but the bottom strand will also undergo processing. Red stars represent interactions that stimulate Artemis activity. Red squares represent known protein-protein binding interactions. Ku interacts with LIV at the region containing the two BRCT domains (41). The region between the BRCT domains of LIV interacts with the helical domain of X4 (7–9). The N-terminal head domain of XLF interacts with the N-terminal head domain of X4 (10). The C terminus of PAXX interacts with Ku (11). The N-terminal BRCT domain of Pol μ interacts with the Ku·DNA complex (19). The FAT domain in DNA-PK_cs interacts with Ku (42). Artemis is activated by its interaction with DNA-PK_cs through its C terminus (amino acids 402- 403). The C terminus of Artemis (amino acids 485–495) interacts with the N-terminal head domain of LIV (20, 21, 31), and the current study indicates that this stimulates Artemis activity (Fig. 2).