DNA repair ͉ nonhomologous end-joining ͉ V(D)J recombinationT he nonhomologous end-joining (NHEJ) pathway is conserved in eukaryotes, from yeast to humans. Without requiring homologous DNA, NHEJ repairs DNA double-strand breaks produced by xenobiotic agents, such as topoisomerase II inhibitors and ionizing radiation, or by the cellular pathway for V(D)J recombination of the immunoglobulin genes (1). Even when the structure of the DNA ends prevents ligation, NHEJ processes the ends and repairs the breaks with high efficiency and minimal nucleotide loss. For ligatable ends, such as the blunt signal ends created by V(D)J recombination, NHEJ suppresses processing and repairs the breaks directly. Thus, NHEJ optimizes the preservation of DNA sequence, but the mechanism is not understood.

Nonhomologous end-joining (NHEJ) repairs DNA doublestrand breaks created by ionizing radiation and V(D)J recombination. To repair the broken ends, NHEJ processes noncompatible ends into a ligatable form but suppresses processing of compatible ends. It is not known how NHEJ controls polymerase and nuclease activities to act exclusively on noncompatible ends. Here, we analyzed processing independently of ligation by using a two-stage assay with extracts that recapitulated the properties of NHEJ in vivo. Processing of noncompatible ends required wortmannin-sensitive kinase activity. Since DNA-dependent protein kinase catalytic subunit (DNA-PKcs) brings the ends together before undergoing activation of its kinase, this suggests that processing occurred after synapsis of the ends. Surprisingly, all polymerase and most nuclease activity required XRCC4/Ligase IV. This suggests a mechanism for how NHEJ suppresses processing to optimize the preservation of DNA sequence. Nonhomologous end-joining (NHEJ)2 preserves chromosomal integrity by repairing DNA double-strand breaks. Processing of DNA ends is a key step in NHEJ since double-strand breaks often create ends that are not directly ligatable. For example, ionizing radiation generates nucleotides with aberrant structures such as 3Ј-phosphate or 3Ј-phosphoglycolate groups, which must be removed by nuclease activity before ligation can occur.Processing of DNA ends in vivo has been studied in the context of V(D)J recombination. The RAG1/RAG2 dimer cleaves two sites in the immunoglobulin locus to create two hairpin coding ends and two blunt signal ends (1). Nuclease activity opens the hairpin ends to leave 3Ј overhangs, which are filled in to generate P-nucleotide addition (2). To fill in 3Ј overhangs, a polymerase must synthesize DNA from a primer on the opposing end. Nucleotide deletion also occurs, but nuclease activity is limited to less than 20 nucleotides, often back to regions of microhomology (3, 4). Strikingly, the blunt signal ends are joined without processing. Thus, the NHEJ reaction is strongly biased toward the preservation of DNA sequence, suppressing processing if it is not needed and limiting the extent of processing when it is required.The NHEJ reaction requires core proteins and processing enzymes (5). The core proteins include Ku, DNA-PKcs, XRCC4/Ligase IV (XL), and Cernunnos (also named XRCC4-like factor, XLF) (6, 7). Proposed processing enzymes include the nuclease Artemis (8) and DNA polymerases and (9 -12). The biochemical properties of these proteins suggest that NHEJ repairs double-strand breaks in an ordered series of steps. Ku binds to DNA ends and translocates inward, recruiting DNAPKcs (13, 14). DNA-PKcs brings the ends together, activating its kinase activity (15). DNA-PKcs then phosphorylates itself and its target proteins (16). The ends are processed if necessary, and XL catalyzes the final ligation step (17).Important questions remain unanswered. NHEJ requires DNA-PKcs kinase activity (18), but it is not known whether the kinase activity aff...