Nonhomologous end-joining is a model for rejoining ends with no homology. Nonhomologous end-joining (NHEJ) is used to repair double-strand breaks where no homology or limited “microhomology” of 1 to 10 nu- cleotides is present. It is used primarily in the G, phase of the cell cycle when there is no closely associated homologous sequence such as the sister chromatid, which is present after DNA replication. Nonhomolo- gous end-joining is sometimes called illegitimate recombination be- cause no homology is used in end joining. The Ku protein complex, a heterodimer consisting ofa 70 and 80 kDa polypeptide, plays an essen- tial role in nonhomologous end-joining. The protein was first discov- ered in the cell nucleus as a target of autoantibodies in patients with scleroderma, an autoimmune disease. (The name “Ku” was derived from the patient's name). The Ku7) and Ku80 subunits are defective in x-ray-sensitive mammalian cells with mutations in the XRCC6 and XRCCS genes, respectively, Homologous genes are present in a wide variety of organisms including yeast and bacteria. The nonhomologous end-joining pathway begins with the binding of the Ku70 80 complex to the broken DNA ends (FIGURE 11.41). In humans, another protein called DNA-dependent protein kinase (DNA-PK) is essential in nonho- mologous end-joining. The Ku complex binds to the ends as a barrel ring structure, The two ends bound by the Ku complex are brought to- gether, assisted. by the MRX (Mre11* Rad50* Xrs2) complex of yeast or the MRN (Mrel1*Rad50*Nbs1) complex in humans, which forms a bridge between the two ends. The ends are then sealed by a specialized ligase called DINA ligase IV. Ligase IV is associated with an- other protein called Lif in yeast and XRCC4 in humans. Other pro- reins, listed in Table 11.3, complete the reaction, Nie proteins , : : J Step in NHEJ S. cerevisiae protein) Mammalian protein | End binding and Kur eo Ku7Qyeo | Liuaapostion | Mre1/Rads0/xrs2__|_DINA-PKes_ | End joining without Dnia DMA, ligase I'v | processing Lift xRCC4 Nei ; XLF End joining with Dri | DNA ligase I'v | processing Lifl ) xACCS | | Mejt Cemunnos/XLF | | Artamis | | _ Palynucleotide kinase (PNK) Pold POLL POLM(a) i Sgrrnnernrcrrnt TL. Ku heterodimers _ = hos | Ku bridiges ends: i i =! Pe LULL LLL —— xACC4LIgIV Processing enzymes fill gap {not shown), XROC4/LigI¥ are recruted (d) ES DNA strands are repaired h ELD FIGURE 11.41 Nonhomologous end-joining. (a) The blue dot on one of the two double-strand break ends signifies a nonligatable and. (b)The double- strand break ends are bound by the Ku heterodimer. (c) The KueDNA complexes are juxtaposed to bridge the ends and the gap is filled in by pracessing enzymes and POLL or POLM. (d) The ends are ligated by the specialized DNA ligase LiglV with its partner XACC4 to (e) repair the double strand break. Adapted from Jones, J.M., Gellert, M., and Yang, W.. Structure 9 (2001): 881-884. Occasionally the broken ends are “dirty” because they lack either a 5"-phosphate end or a 3’-hydroxy end and so cannot serve as a sub- strate for DNA ligase. “Dirty” ends are produced by radiation, oxida- tion, and radiomimetics (chemicals thar make double-strand breaks) or by enzymes such as nucleases and defective topoisomerases. Additional processing is required to convert the “dirty” ends into ends that can be joined together by DNA ligase. This processing can lead to small mu- tations consisting of a few nucleotides of insertion or deletion at the end joints (FIGURE 11.42). The nonhomologous end-joining process is related to the mecha- nism of DNA transposition, discussed in Chapter 12. It is also relatedTT Swnole religation Doebebebenhsaletandentanl oss ' Precise aa a ' Multiple damaged sites Radiation FRackomimetics —_—_— . r Oxidation ee 2 Enzymatic degradation — — a Physcial stress: q : ' Endonucleases 1 Processed f ‘ , Free DSB ends ' eats op Replication —— ‘ ' Recessed —oOo FIGURE 11.42 Nonhomologous end-joining of dirty ends. Many double-strand break ends are damaged (described in the left panel) and cannot engage in a simple ligation reaction, These must be processed first ta remove the damaged ends. In other cases, the ends require a few nucleotides to be added or removed to allow annealing of two to three nucleotides prior to liga- tion, Adapted from Daley, J. M,, etal, Anau. Rev, Genet, 39 (2005): 431-451. to V(D)J recombination, which is the process of making a mature immunoglobulin gene from separated modular parts, V(D)J recombi nation, discussed later in this chapter, uses some of the core nonho- mologous end-joining factors in addition to novel factors specific for V(D)J recombination.