nonhomologous end joining (NHEJ) is the major pathway for repair of DNA double-strand breaks (DSBs) in human Disopyramide cells. of the core protein components for NHEJ: Ku70/Ku80 heterodimer; the DNA dependent protein kinase catalytic subunit (DNA-PKcs); the structure-specific endonuclease Artemis along with polynucleotide kinase/phosphatase (PNKP) aprataxin and PNKP related protein (APLF); the scaffolding proteins XRCC4 and XLF (XRCC4-like factor); DNA polymerases and DNA ligase IV (Lig IV). The dynamic assembly of multi-protein NHEJ complexes at DSBs is regulated in part by protein phosphorylation. The basic steps of NHEJ have been biochemically defined to require: 1) DSB detection by the Ku heterodimer with subsequent DNA-PKcs tethering to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK) 2 lesion processing and 3) DNA end ligation by Lig IV which functions in complex with XRCC4 and XLF. The current integration of structures by combined methods is resolving puzzles regarding the mechanisms coordination and regulation of these three basic steps. Overall structural results suggest the NHEJ system forms a flexing Disopyramide scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular springs suitable to store and Disopyramide release conformational energy to apply forces to regulate NHEJ complexes and the DNA substrate for DNA end protection processing and ligation. 1 Introduction nonhomologous end joining (NHEJ sometimes referred to as classical or C- NHEJ) is the major pathway for repair of ionizing Disopyramide radiation (IR)-induced double-stranded DNA breaks (DSBs) in human cells [1 2 NHEJ is required for antigen receptor gene rearrangements via V(D)J recombination and the development of T and B cells in the vertebrate immune Disopyramide system [3] and it is implicated in both the generation and prevention of non-homologous chromosomal translocations a major hallmark of genomic instability and many human cancers [4]. The main proteins required for NHEJ are the Ku70/Ku80 heterodimer the catalytic subunit of the Disopyramide DNA-dependent protein kinase (DNA-PKcs) the endo/structure specific nuclease Artemis the scaffolding protein XRCC4 DNA ligase IV (Lig IV) and XRCC4-like factor XLF. NHEJ can be thought of as occurring in three distinct stages: (1) detection of the DSB by the Ku heterodimer SSI-2 with subsequent tethering by DNA-PKcs to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK) (2) processing of IR-induced lesions and (3) ligation of the DNA ends by Lig IV that functions in complex with XRCC4 and XLF. DNA end processing is required to remove damaged DNA and non-ligatable end groups at the termini of the DSB to facilitate ligation. End processing in response to IR-induced damage may include a variety of enzymes including polynucleotide kinase/phosphatase (PNKP) aprataxin and PNKP related protein (APLF) DNA polymerases and Artemis [1 5 6 (Fig. 1). Figure 1 A model for non-homologous end joining Over the past few years it has become apparent that Ku plays multiple roles in NHEJ; not only does it recognize DSB ends it has catalytic activity [7 8 and is required for recruitment of multiple NHEJ proteins to the DSB (reviewed in [5 6 A picture is thus emerging of the dynamic assembly of a multi-protein NHEJ complex at DSBs the function of which is regulated at least in part by protein phosphorylation [9]. Unraveling the multiple protein-protein and protein-DNA interactions within such dynamic protein-DNA assemblies presents formidable challenges for traditional approaches such as X-ray crystallography; however small angle X-ray scattering (SAXS) has emerged as a powerful tool for elucidating overall shapes and conformations of protein-protein and protein-DNA complexes [10 11 In addition we are learning more about the pathway and cross-pathway interactions between NHEJ and homologous recombination (HR) and its initiating complex MRE11-RAD50-NBS1 (MRN) [12]. During S and G2 phases of the cell cycle when NHEJ and HR are both active the choice between which of the two pathways is used for repair is controlled in part by resection of DSBs as the 3′ single-strand DNA (ssDNA) generated by extensive resection inhibits NHEJ but is required for Rad51 filament formation and strand invasion during HR [13]. We can now start to consider the structural implications of the complexes acting in the two DSB repair pathways for mechanisms of pathway choice. Here we will review how structural biology on individual NHEJ components and in some cases.