REVIEW Non-homologousend-joining,astickyaffair DC van Gent 1 and M van der Burg 2 1 Department of Cell Biology and Genetics, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands and 2 Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands Rejoining of broken chromosomes is crucial for cell survivalandpreventionofmalignanttransformation.Most mammalian cells rely primarily on the non-homologous end-joining pathway of DNA double-strand break (DSB) repairtoaccomplishthistask.Thisreviewfocusesbothon the core non-homologous end-joining machinery, which consists of DNA-dependent protein kinase and the ligase IV/XRCC4 complex, and on accessory factors that facilitate rejoining of a subset of the DSBs. We discuss howtheATMproteinkinaseandtheMre11/Rad50/Nbs1 complex might function in DSB repair and what role ionizingradiation-inducedfocimayplayinthisprocess. Oncogene (2007) 26, 7731–7740; doi:10.1038/sj.onc.1210871 Keywords: DNA repair; V(D)J recombination; DNA double-strand break repair; severe combined immuno- deficiency; DNA damage response; chromosomal instability TheDNAdamageresponse DNA damage induces several responses, including DNA repair, cell cycle checkpoints and apoptosis (Zhou and Elledge, 2000; Bartek and Lukas, 2007). This is collectively referred to as the DNA damage response (DDR). DNA double-strand breaks (DSBs) pose a particularly severe threat to genome stability, since lack of repair may cause loss of chromosome fragments during mitosis, whereas coupling of the wrong DNA ends is the basis for chromosomal translocations that can induce neoplastic transformation (Burma et al., 2006). To prevent loss of genetic material during mitosis, cells halt their cell cycle at the G 2 /M boundary until the majority of DSBs is repaired. On the other hand, excessively damaged cells can be eliminated by activation of apoptotic pathways, thereby preventing survival of cells that might have lost proper growth regulation, for example by loss of heterozygosity of a tumor suppressor gene. The most important factor for cell survival is repair of the DNA damage. DSBs can be repaired by two fundamentally different mechanisms (Helleday et al., 2007). Homologous recombination uses the information on the sister chromatid or the homologous chromosome to align both DNA ends. In mammalian cells, this repair pathway is probably restricted to the S and G 2 phases of the cell cycle when a sister chromatid is present. Non- homologous end-joining (NHEJ) does not require a homologous DNA template, although short patches of homologous nucleotides (microhomologies of one or a few base pairs) are frequently used to align the DNA ends. This review focuses on the mechanism of NHEJ and the implications of defects in this repair pathway. OutlineoftheNHEJpathway The NHEJ reaction starts with binding of the Ku70/80 heterodimer to a DNA end (Figure 1) (Weterings and van Gent, 2004). DNA end bound Ku70/80 then attracts the catalytic subunit of the DNA-dependent protein kinase (DNA-PK CS ) and activates its protein kinase activity (Smith and Jackson, 1999). Although many different targets have been identified, the major function for this phosphorylation activity appears to be regulation of the NHEJ reaction by DNA-PK CS autophosphoryla- tion (Chan etal., 2002; Ding etal., 2003; Weterings etal., 2003). This reaction takes place after juxtaposition of DNA ends and is required for proper regulation of DNA end accessibility for other NHEJ proteins (Meek et al., 2007). Compatible DNA ends can now be joined directly by the ligase IV/XRCC4 complex. This reaction is stimulated by the recently discovered XLF/Cernunnos protein, which interacts with XRCC4 (Ahnesorg et al., 2006; Buck et al., 2006). However, in many circum- stances the DNA ends are not compatible. Ionizing radiation, for example, creates a large number of ends that contain damaged bases and/or DNA backbone sugars that need processing before ligation. Therefore, NHEJ can accommodate nucleases, DNA polymerases polynucleotide kinase and other enzymes that render such ends ligatable by the ligase IV/XRCC4 complex. NHEJ is not only important for repair of DSBs that result from exogenous and endogenous DNA damaging agent, but also for repair of DSBs generated during V(D)J recombination, which takes place during B- and T-cell differentiation to generate antigen specific recep- tors (Jung et al., 2006). Variable (V), Diversity (D) and Joining (J) segments in Immunoglobulin (Ig) and T Cell Receptor (TCR) genes are joined to form the mature Correspondence: Dr DC van Gent, Department of Cell Biology and Genetics, Erasmus MC, PO Box 2040, Rotterdam 3000 CA, The Netherlands. E-mail: d.vangent@erasmusmc.nl Oncogene (2007) 26, 7731–7740 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc