The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway

Abstract

Double-strand DNA breaks are common events in eukaryotic cells, and there are two major pathways for repairing them: homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). The various causes of double-strand breaks (DSBs) result in a diverse chemistry of DNA ends that must be repaired. Across NHEJ evolution, the enzymes of the NHEJ pathway exhibit a remarkable degree of structural tolerance in the range of DNA end substrate configurations upon which they can act. In vertebrate cells, the nuclease, DNA polymerases, and ligase of NHEJ are the most mechanistically flexible and multifunctional enzymes in each of their classes. Unlike repair pathways for more defined lesions, NHEJ repair enzymes act iteratively, act in any order, and can function independently of one another at each of the two DNA ends being joined. NHEJ is critical not only for the repair of pathologic DSBs as in chromosomal translocations, but also for the repair of physiologic DSBs created during variable (diversity) joining [V(D)J] recombination and class switch recombination (CSR). Therefore, patients lacking normal NHEJ are not only sensitive to ionizing radiation (IR), but also severely immunodeficient.

Figure 1. Causes and Repair of Double-Strand DNA Breaks

Physiologic and pathologic causes of double-strand breaks in mammalian somatic cells are listed at the top. During S and G2 of the cell cycle, homology-directed repair is common because the two sister chromatids are in close proximity, providing a nearby homology donor. Homology-directed repair includes homologous recombination (HR) and single-strand annealing (SSA). At any time in the cell cycle, double-strand breaks can be repaired by nonhomologous DNA end joining (NHEJ). Proteins involved in the repair pathways are listed.

Figure 2. General Steps of Nonhomologous DNA End Joining

Ku binding to the DNA ends at a DSB improves binding by nuclease, polymerase and ligase components. Flexibility in the loading of these enzymatic components, the option to load repeatedly (iteratively), and independent processing of the two DNA end all permit mechanistic flexibility for the NHEJ process. This mechanistic flexibility is essential to permit NHEJ to handle a very diverse array of DSB end configurations and to join them. In addition to the overall mechanistic flexibility, each component exhibits enzymatic flexibility and multifunctionality, as discussed in the text.

Figure 3. Interactions Between NHEJ Proteins

Physical interactions between NHEJ components are summarized. In addition, interactions between XRCC4 and DNA-PKcs are discussed in the text, as are functional interactions.

Figure 4. Diagrams of Domains within NHEJ Proteins

a. Ku is a heterodimer of Ku70 and 86. vWA designates von Willebrand domains. SAP designates a SAF-A/B, Acinus, and PIAS domain and may be involved in DNA binding. b. DNA-PKcs autophosphorylation sites are shown in red (90, 95, 96). The function of each phosphorylation site (A-E, L, M, P-R) and cluster (N and JK) are still under study. Adjacent phosphorylation sites that are linked by a bracket have not been functionally dissected from one another. LRR designates the leucine-rich region. The FAT-C domain is a FAT domain at the C-terminus. PI3K designates the PI3 kinase domain. PRD designates the PI3K regulatory domain. c. Artemis is phosphorylated by DNA-PKcs at 11 sites within the C-terminal portion (green) (162, 163). Amino acids 156 to 385 share conserved sequence with those metallo-β-lactamases that act on nucleic acids (164). This region has been called the β-CASP domain (metallo-β-lactamase-associated CPSF Artemis SNM1 PSO2) (165). d. POL × polymerase family. Pol mu and pol lambda are involved in NHEJ in mammalian somatic cells generally. TdT is only expressed in early lymphoid cells where it participates in NHEJ primarily in the context of V(D)J recombination. e. The NHEJ ligase complex consists of XLF (Cernunnos), XRCC4, DNA ligase IV. The red arrows indicate the regions of physical interaction (118, 119). OBD in ligase IV is the oligo-binding domain, and AdB is the adenylation domain. f. Polynucleotide kinase (PNK), Aprataxin (APTX), and PALF (APLF) are ancillary components that bind to XRCC4 of the ligase complex. The PBZ domain appears to be important for PARP-1 binding, for poly-ADPribose binding, and for nuclease activity. FHA designates the forkhead-associated domain.