DNA-PK: A synopsis beyond synapsis.

Given its central role in life, DNA is remarkably easy to damage. Double strand breaks (DSBs) are the most toxic form of DNA damage, and DSBs pose the greatest danger to genomic integrity. In higher vertebrates, the non-homologous end joining pathway (NHEJ) is the predominate pathway that repairs DSBs. NHEJ has three steps: 1) DNA end recognition by the DNA dependent protein kinase [DNA-PK], 2) DNA end-processing by numerous NHEJ accessory factors, and 3) DNA end ligation by the DNA ligase IV complex (LX4). Although this would appear to be a relatively simple mechanism, it has become increasingly apparent that it is not. Recently, much insight has been derived regarding the mechanism of non-homologous end joining through a proliferation of cryo-EM studies, structure-function mutational experiments informed by these new structural data, and novel single-molecule imaging approaches. An emerging consensus in the field is that NHEJ progresses from initial DSB end recognition by DNA-PK to synapsis of the two DNA ends in a long-range synaptic complex where ends are held too far apart (115 Å) for ligation, and then progress to a short-range synaptic complex where ends are positioned close enough for ligation. What was surprising from these structural studies was the observation of two distinct types of DNA-PK dimers that represent NHEJ long-range complexes. In this review, we summarize current knowledge about the function of the distinct NHEJ synaptic complexes and align this new information with emerging cellular single-molecule microscopy studies as well as with previous studies of DNA-PK's function in repair.

Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells.

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-stranded breaks (DSBs) in vertebrates. However, due to challenges in detecting DSBs in living cells, the repair capacity of the NHEJ pathway is unknown. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed ~115Å apart in one of several long-range synaptic complexes before transitioning into a short-range synaptic complex that juxtaposes DNA ends to facilitate ligation. The transition from long-range to short-range synaptic complexes involves both conformational and compositional changes of the NHEJ factors bound to the DNA break. Importantly, it is unclear how NHEJ proceeds in vivo because of the challenges involved in analyzing recruitment of NHEJ factors to DSBs over time in living cells. Here, we develop a new approach to study the temporal and compositional dynamics of NHEJ complexes using live cell single-molecule imaging. Our results provide direct evidence for stepwise maturation of the NHEJ complex, pinpoint key regulatory steps in NHEJ progression, and define the overall repair capacity NHEJ in living cells.

Two distinct long-range synaptic complexes promote different aspects of end processing prior to repair of DNA breaks by non-homologous end joining.

Non-homologous end joining is the major double-strand break repair (DSBR) pathway in mammals. DNA-PK is the hub and organizer of multiple steps in non-homologous end joining (NHEJ). Recent high-resolution structures show how two distinct NHEJ complexes "synapse" two DNA ends. One complex includes a DNA-PK dimer mediated by XLF, whereas a distinct DNA-PK dimer forms via a domain-swap mechanism where the C terminus of Ku80 from one DNA-PK protomer interacts with another DNA-PK protomer in trans. Remarkably, the distance between the two synapsed DNA ends in both dimers is the same (∼115 Å), which matches the distance observed in the initial description of an NHEJ long-range synaptic complex. Here, a mutational strategy is used to demonstrate distinct cellular function(s) of the two dimers: one promoting fill-in end processing, while the other promotes DNA end resection. Thus, the specific DNA-PK dimer formed (which may be impacted by DNA end structure) dictates the mechanism by which ends will be made ligatable.

Unravelling the complexities of DNA-PK activation by structure-based mutagenesis.

It has been known for decades that the DNA-dependent protein kinase (DNA-PK) is only an active serine/threonine protein kinase when it is bound to a DNA double-stranded end; still, the molecular details of how this activation is achieved have remained elusive. The recent surge in structural information for DNA-PK complexes has provided valuable insights into the process of DNA end recognition by DNA-PK. A particularly intriguing feature of this kinase is a region of the protein that can transition from a seemingly structurally disordered state to a single alpha-helix that traverses down the DNA binding cradle. The DNA-PK bound DNA end of the DNA substrate engages with and appears to split around this helix which has been named the DNA End Blocking helix (DEB). Here a mutational approach is utilized to clarify the role of the DEB, and how DNA ends activate the enzyme. Our data suggest two distinct methods of kinase activation that is dependent on the DNA end chemistry. If the DNA end can split around the helix and stabilize the interaction between the DNA end and the DEB with a recently defined Helix-Hairpin-Helix (HHH) motif, the kinase forms an end-protection monomer that is active towards DNA-PK's many substrates. But if the DNA end cannot stably interact with the DEB [because of the DNA end structure, for instance hairpins, or because the DEB has been disrupted by mutation], the kinase is only partially activated, resulting in specific autophosphorylations of the DNA-PK monomer that allows nucleolytic end-processing. We posit that mutants that disrupt the capacity to stably generate the DEB/HHH DNA end-interaction are inefficient in generating the dimer complex that is requisite for NHEJ. In support of this idea, mutations that promote formation of this dimer partially rescue the severe cellular phenotypes associated with mutation of the DEB helix.

Catalytically inactive DNA ligase IV promotes DNA repair in living cells.

DNA double strand breaks (DSBs) are induced by external genotoxic agents (ionizing radiation or genotoxins) or by internal processes (recombination intermediates in lymphocytes or by replication errors). The DNA ends induced by these genotoxic processes are often not ligatable, requiring potentially mutagenic end-processing to render ends compatible for ligation by non-homologous end-joining (NHEJ). Using single molecule approaches, Loparo et al. propose that NHEJ fidelity can be maintained by restricting end-processing to a ligation competent short-range NHEJ complex that 'maximizes the fidelity of DNA repair'. These in vitro studies show that although this short-range NHEJ complex requires DNA ligase IV (Lig4), its catalytic activity is dispensable. Here using cellular models, we show that inactive Lig4 robustly promotes DNA repair in living cells. Compared to repair products from wild-type cells, those isolated from cells with inactive Lig4 show a somewhat increased fraction that utilize micro-homology (MH) at the joining site consistent with alternative end-joining (a-EJ). But unlike a-EJ in the absence of NHEJ, a large percentage of joints isolated from cells with inactive Lig4 occur with no MH - thus, clearly distinct from a-EJ. Finally, biochemical assays demonstrate that the inactive Lig4 complex promotes the activity of DNA ligase III (Lig3).

Autophosphorylation transforms DNA-PK from protecting to processing DNA ends.

The DNA-dependent protein kinase (DNA-PK) initially protects broken DNA ends but then promotes their processing during non-homologous end joining (NHEJ). Before ligation by NHEJ, DNA hairpin ends generated during V(D)J recombination must be opened by the Artemis nuclease, together with autophosphorylated DNA-PK. Structures of DNA-PK bound to DNA before and after phosphorylation, and in complex with Artemis and a DNA hairpin, reveal an essential functional switch. When bound to open DNA ends in its protection mode, DNA-PK is inhibited for cis-autophosphorylation of the so-called ABCDE cluster but activated for phosphorylation of other targets. In contrast, DNA hairpin ends promote cis-autophosphorylation. Phosphorylation of four Thr residues in ABCDE leads to gross structural rearrangement of DNA-PK, widening the DNA binding groove for Artemis recruitment and hairpin cleavage. Meanwhile, Artemis locks DNA-PK into the kinase-inactive state. Kinase activity and autophosphorylation of DNA-PK are regulated by different DNA ends, feeding forward to coordinate NHEJ events.

Cryo-EM of NHEJ supercomplexes provides insights into DNA repair.

Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.