The molecular basis and disease relevance of non-homologous DNA end joining

Abstract

Non-homologous DNA end joining (NHEJ) is the predominant repair mechanism of any type of DNA double-strand break (DSB) during most of the cell cycle and is essential for the development of antigen receptors. Defects in NHEJ result in sensitivity to ionizing radiation and loss of lymphocytes. The most critical step of NHEJ is synapsis, or the juxtaposition of the two DNA ends of a DSB, because all subsequent steps rely on it. Recent findings show that, like the end processing step, synapsis can be achieved through several mechanisms. In this Review, we first discuss repair pathway choice between NHEJ and other DSB repair pathways. We then integrate recent insights into the mechanisms of NHEJ synapsis with updates on other steps of NHEJ, such as DNA end processing and ligation. Finally, we discuss NHEJ-related human diseases, including inherited disorders and neoplasia, which arise from rare failures at different NHEJ steps.

Fig. 1 |. Overview of the non-homologous DNA end joining process.

a | Non-homologous DNA end joining (NHEJ) begins with binding of the Ku70–Ku80 heterodimer to the ends of the double-strand break (DSB). The biochemical steps of NHEJ include synapsis, which brings the diffused DNA ends back into proximity, end processing and finally ligation. Two independent mechanisms exist for NHEJ synapsis. One depends on Ku70–Ku80, XRCC4–DNA ligase4 (LIG4), XRCC4-like factor (XLF) and/or paralogue of XRCC4 and XLF (PAXX). The other depends on DNA polymerase-μ (Polμ). Synapsis (pink box) is depicted in detail in FIG. 3. DNA ends that are incompatible for direct ligation by LIG4 are processed by the nuclease Artemis or by polymerases (Polμ, Polλ and terminal deoxynucleotidyl transferase (TdT)) to become compatible for ligation. Artemis and tyrosyl-DNA phosphodiesterase 1 (TDP1) can remove 3′-phosphoglycolates (not shown), which block ligation and can be generated at DSBs caused by ionizing radiation (IR). End processing (lilac box) is presented in detail in FIG. 4. Naturally occurring DSBs almost always feature sequence alterations at the DNA ends, even before their modification by NHEJ factors (blue box). Together, the diverse nature of the damage ends and of end processing give rise to diverse repair junctions, including small deletions and insertions, although precisely joined products are also found at low frequency, especially when the ends are compatible for direct ligation^,. The green lines represent added nucleotides. b | The end joining process is flexible and iterative, meaning that DNA ends with diverse configurations can be covalently ligated following various modifications. XRCC4–LIG4 can ligate each strand independently of the other. The Artemis–DNA-dependent protein kinase catalytic subunit (DNA-PKcs) complex can trim overhangs to expose complementary regions and can also nick a gap at the ligated strand. The nicking of the ligated strand would generate the same or modified DNA ends, possibly with overhangs for another round of end joining. DNA polymerases (Polμ and Polλ) can add nucleotides to either create microhomologies or to fill in gaps to facilitate DNA strand ligation. Nucleotide addition by polymerases may also generate a flap (not shown), which requires endonucleolytic cleavage by Artemis. The iterative nature of NHEJ allows multiple rounds of revision. PNKP, polynucleotide kinase 3′-phosphatase.

Fig. 2 |. DSB repair pathway choice.

DNA double-strand breaks (DSBs) are repaired by non-homologous DNA end joining (NHEJ), alternative end joining (a-EJ), single-strand annealing (SSA) and homologous recombination (HR). NHEJ is the predominant DSB repair pathway (bold arrow). Pathway choice is largely dictated by the availability of homology (called microhomology if the length is less than 20 bp) between the DNA end overhangs. NHEJ requires either no microhomology or, more often, 1–4 bp of terminal microhomology. a-EJ typically requires microhomology of at least 2 bp (usually more) and less than 20 bp. SSA requires homology of typically more than 50 bp, and the homology requirement for HR is typically more than 100 bp. The exposure of terminal (micro)homology is partly determined by the extent of DNA end protection versus nucleolytic resection. TP53-binding protein 1 (53BP1) and its effectors, RAP1-interacting factor 1 (RIF1), the shieldin complex (comprising shieldin complex subunit 1 (SHLD1), SHLD2, SHLD3 and revertibility protein 7 homologue (REV7)), the conserved telomere maintenance component 1 (CTC1)–oligonucleotide/oligosaccharide-binding fold-containing protein 1 (STN1)–telomere length regulation protein TEN1 homologue (TEN1) (CST) complex and polymerase-α (Polα) protect the ends from extensive resection. By contrast, CtBP-interacting protein (CtIP) and the MRE11–RAD50–NBS1 (MRN) endonuclease first nick one strand near the 5′ end and then degrade the strand in a 3′ to 5′ direction towards the end, thereby creating a short 3′ overhang, which is suitable for a-EJ. Poly(ADP-ribose) polymerase 1 (PARP1) and Polθ are important for a-EJ. The nucleases Bloom syndrome protein (BLM)–DNA replication ATP-dependent helicase/nuclease DNA2 and exonuclease 1 (EXO1) can mediate longer resection in a 5′ to 3′ manner, and replication protein A (RPA) protects the resulting single-stranded DNA (ssDNA) to facilitate HR and SSA. Annealing of homologous sequences by RAD52 is important for SSA, and the 3′ non-homologous ssDNA flaps are cut by XPF–ERCC1 before ligation by DNA ligase 1 (LIG1). RAD51, breast cancer type 1 susceptibility protein (BRCA1), BRCA2 and RAD54 are essential to promote HR. The (micro)homology regions within the repair products are labelled with colour; the proteins highlighted with colour are those essential for the corresponding pathways. DNA-PKcs, DNA-dependent protein kinase catalytic subunit; PAXX, paralogue of XRCC4 and XLF; XLF, XRCC4-like factor.

Fig. 3 |. Mechanisms of NHEJ synapsis.

At least two mechanisms exist for non-homologous DNA end joining (NHEJ) synapsis: a Ku70–Ku80–XRCC4–DNA ligase 4 (LIG4)–XRCC4-like factor (XLF)-dependent mechanism and a DNA polymerase-μ (Polμ)-dependent mechanism. The choice of synapsis mechanism depends on the configurations of the DNA ends and the availability of different NHEJ proteins. a | Single-molecule Förster resonance energy transfer for synapsis analysis. A fluorescently labelled DNA molecule is immobilized on a slide, and a differently labelled DNA molecule together with NHEJ proteins is then added onto the slide to initiate synapsis. b | The Ku70–Ku80–XRCC4–LIG4–XLF-dependent mechanism of synapsis. Two structurally different synaptic complexes corresponding to flexible synapsis and close synapsis are formed through this mechanism. In flexible synapsis, the two duplexes are laterally aligned; flexible synapsis can be mediated by Ku70–Ku80 and XRCC4–LIG4 for both blunt ends and overhangs. XLF and/or paralogue of XRCC4 and XLF (PAXX) can promote the close synapsis in either a stepwise manner, in which they drive the two duplexes from the lateral configuration (flexible synapsis) to an end-to-end close contact configuration, or in a single step, in which the close synapsis is immediately formed by Ku70–Ku80, XRCC4–LIG4 and XLF or PAXX. When short terminal microhomologies exist between the overhangs, Ku70–Ku80 and XRCC4–LIG4 can also promote close synapsis in the absence of XLF and PAXX (not shown). The two duplexes within the close synapsis can be readily ligated by XRCC4–LIG4. c | The Polμ-dependent mechanism of synapsis. Close synapsis of DNA ends with 3′ overhangs and short microhomology can be mediated by Polμ. Nucleotide addition can then occur within the close synapsis. High abundance of Ku70–Ku80 can inhibit Polμ-mediated synapsis if Ku70–Ku80 occupies the DNA ends first. XRCC4–LIG4 can reverse this inhibition, possibly by pushing Ku70–Ku80 inwards along the DNA, thereby exposing overhangs and helping recruit Polμ to mediate NHEJ. Not shown are the 5′ overhang configuration, because it can be either easily trimmed by Artemis or filled in by Polμ or Polλ to generate a blunt end; filament formation — for chromatinized DNA, filaments might be important for synapsis; and a suggested role for DNA-dependent protein kinase catalytic subunit in synapsis^– (Supplementary Box 1). dNTP, deoxyribonucleoside triphosphate. Parts a and b adapted from REF., Springer Nature.

Fig. 4 |. Various NHEJ end processing mechanisms.

a | Resection of broken DNA ends with different configurations. aa | Blunt ends are often readily ligated and repaired without processing. ab | At resection-dependent compatible ends, the nuclease Artemis, which interacts with and is activated by DNA-dependent protein kinase catalytic subunit (DNA-PKcs) can cut off the non-base-paired flap to expose the imbedded short microhomology (of ~4 bp). ac | Incompatible 3′ overhang ends are available for iterative processing until a thermodynamically stable junction is achieved through hydrogen bonding across the double-strand break junction. Artemis–DNA-PKcs mediates end resection, and DNA polymerases (Pol) add nucleotides to the ends, thereby generating short microhomologies between ends (orange). ad | Incompatible 5′ overhang ends can be readily trimmed by Artemis–DNA-PKcs or filled-in by DNA polymerases to generate blunt ends. B | Polymerase activity at different end configurations. Ba | Polymerase-μ (Polμ) and terminal deoxynucleotidyl transferase (TdT) can add nucleotides to a blunt end in a template-independent manner. Bb | Polλ and Polμ can fill in gaps at 3′ recessed DNA ends. Bc | Polλ and Polμ can add nucleotides to the blunt end in a template-dependent manner. The preferentially added nucleotides are complementary to the terminal bases at the other DNA end. Bd | Polλ and polμ can fill in gaps at junctions. Be | Polμ, TdT and Polλ can perform templated in trans synthesis for overhangs with short regions of terminal base pairing; that is, the polymerases can use a 3′ overhang of another DNA end as a template for nucleotide addition. Polμ and TdT have higher activity than Polλ in this context. Bf | Pol μ and TdT can add nucleotides to 3′ non-complementary overhangs in a template-dependent manner. Bg | The 3′ primer end (light blue) can slip inwards, followed by synthesis by Polμ and Polλ, leading to the generation of direct repeats, which are found at some non-homologous DNA end joining (NHEJ) repair junctions. Polλ may have higher activity of generating repeats than Polμ. Bh | When Polμ or TdT adds nucleotides in a template-independent manner, the newly generated overhang may fold back and allow continued synthesis from the same strand end by Polμ or Polλ. The template-independent addition and then fold-back synthesis can generate inverted repeats at NHEJ junctions. The orange lines represent added nucleotides.

Fig. 5 |. Disease-related NHEJ hypomorphic protein variants identified in humans.

Locations of mutations identified in humans giving rise to hypomorphic non-homologous DNA end joining (NHEJ) proteins are shown. The clinical features related to these hypomorphic variants are listed in Supplementary Table 1. Additional Artemis alterations identified in humans are reported elsewhere. Protein domains and their approximate positions are also shown. Blue parts represent protein domains, and grey parts represent linker regions. ‘Δ’ represents a deletion, and ‘X’ denotes a stop codon. The number following ‘X’ denotes the number of amino acids (aa) from the mutation to the stop codon. β-CASP, cleavage and polyadenylation specificity factor domain; ABCDE, DNA-PKcs autophosphorylation cluster spanning residues 2609–2647; BRCT, breast cancer-associated carboxy-terminal domain; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; FAT, FRAP, ATM and TRRAP domain; FAT-C, carboxy-terminal domain of DNA-PKcs; fs, frameshift; ins, insertion; LIG4, DNA ligase 4; PI3K, phosphatidylinositol 3-kinase domain; PQR, DNA-PKcs autophosphorylation sites spanning residues 2023–2056; XID, XRCC4 interaction domain; XLF, XRCC4-like factor.