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The DNA ligase IV-Xrcc4 complex is responsible for the ligation of broken DNA ends in the non-homologous end-joining (NHEJ) pathway of DNA double strand break repair in mammals. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. Only partial structural information for Lig4 and Xrcc4 is available, while the structure of the full-length proteins and their arrangement within the Lig4-Xrcc4 complex is unknown. The C-terminal domain of Xrcc4, whose structure has not been solved, contains phosphorylation sites for DNA-PKcs and is phylogenetically conserved, indicative of a regulatory role in NHEJ. Here, we have purified full length Xrcc4 and the Lig4-Xrcc4 complex, and analysed their structure by single-particle electron microscopy. The three-dimensional structure of Xrcc4 at a resolution of approximately 37A reveals that the C-terminus of Xrcc4 forms a dimeric globular domain connected to the N-terminus by a coiled-coil. The N- and C-terminal domains of Xrcc4 locate at opposite ends of an elongated molecule. The electron microscopy images of the Lig4-Xrcc4 complex were examined by two-dimensional image processing and a double-labelling strategy, identifying the site of the C-terminus of Xrcc4 and the catalytic core of Lig4 within the complex. The catalytic domains of Lig4 were found to be in the vicinity of the N-terminus of Xrcc4. We provide a first sight of the structural organization of the Lig4-Xrcc4 complex, which suggests that the BRCT domains could provide the link of the ligase to Xrcc4 while permitting some movements of the catalytic domains of Lig4. This arrangement may facilitate the ligation of diverse configurations of damaged DNA.

Recognition of DNA double-strand breaks during non-homologous end joining is carried out by the Ku70-Ku80 protein, a 150 kDa heterodimer that recruits the DNA repair kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to the lesion. The atomic structure of a truncated Ku70-Ku80 was determined; however, the subunit-specific carboxy-terminal domain of Ku80--essential for binding to DNA-PKcs--was determined only in isolation, and the C-terminal domain of Ku70 was not resolved in its DNA-bound conformation. Both regions are conserved and mediate protein-protein interactions specific to mammals. Here, we reconstruct the three-dimensional structure of the human full-length Ku70-Ku80 dimer at 25 A resolution, alone and in complex with DNA, by using single-particle electron microscopy. We map the C-terminal regions of both subunits, and their conformational changes after DNA and DNA-PKcs binding to define a molecular model of the functions of these domains during DNA repair in the context of full-length Ku70-Ku80 protein.

DNA-PKcs is a large (approximately 470 kDa) kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). DNA-PKcs is recruited to DSBs by the Ku70/Ku80 heterodimer, with which it forms the core of a multiprotein complex that promotes synapsis of the broken DNA ends. We have purified the human DNA-PKcs/Ku70/Ku80 holoenzyme assembled on a DNA molecule. Its three-dimensional (3D) structure at approximately 25 Angstroms resolution was determined by single-particle electron microscopy. Binding of Ku and DNA elicits conformational changes in the FAT and FATC domains of DNA-PKcs. Dimeric particles are observed in which two DNA-PKcs/Ku70/Ku80 holoenzymes interact through the N-terminal HEAT repeats. The proximity of the dimer contacts to the likely positions of the DNA ends suggests that these represent synaptic complexes that maintain broken DNA ends in proximity and provide a platform for access of the various enzymes required for end processing and ligation.

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is essential for the repair of double-stranded DNA breaks (DSBs) in non- homologous end joining (NHEJ) and during V(D)J recombination. DNA-PKcs binds single- and double-stranded DNA in vitro, and in vivo the Ku heterodimer probably helps recruit it to DSBs with high affinity. Once loaded onto DNA, DNA-PKcs acts as a scaffold for other repair factors to generate a multiprotein complex that brings the two DNA ends together. Human DNA-PKcs has been analysed by electron microscopy in the absence and presence of double-stranded DNA, and the three-dimensional reconstruction of DNA-bound DNA-PKcs displays large conformational changes when compared with the unbound protein. DNA-PKcs seems to use a palm-like domain to clip onto the DNA, and this new conformation correlates with the activation of the kinase. We suggest that the observed domain movements might help the binding and maintenance of DNA-PKcs' interaction with DNA at the sites of damage, and that these conformational changes activate the kinase.

The human tumor suppressor gene ataxia telangiectasia mutated (ATM) encodes a 3056 amino-acid protein kinase that regulates cell cycle checkpoints. ATM is defective in the neurodegenerative and cancer predisposition syndrome ataxia-telangiectasia. ATM protein kinase is activated by DNA damage and responds by phosphorylating downstream effectors involved in cell cycle arrest and DNA repair, such as p53, MDM2, CHEK2, BRCA1 and H2AX. ATM is probably a component of, or in close proximity to, the double-stranded DNA break-sensing machinery. We have observed purified human ATM protein, ATM-DNA and ATM-DNA-avidin bound complexes by single-particle electron microscopy and obtained three-dimensional reconstructions which show that ATM is composed of two main domains comprising a head and an arm. DNA binding to ATM induces a large conformational movement of the arm-like domain. Taken together, these three structures suggest that ATM is capable of interacting with DNA, using its arm to clamp around the double helix.

Detection of cytosolic DNA is a central element of the innate immunity system against viral infection. The Ku heterodimer, a component of the NHEJ pathway of DNA repair in the nucleus, functions as DNA sensor that detects dsDNA of viruses that replicate in the cytoplasm. Vaccinia virus expresses two proteins, C4 and C16, that inactivate DNA sensing and enhance virulence. The structural basis for this is unknown. Here we determine the structure of the C16 – Ku complex using cryoEM. Ku binds dsDNA by a preformed ring but C16 sterically blocks this access route, abrogating binding to a dsDNA end and its insertion into DNA-PK, thereby averting signalling into the downstream innate immunity system. C4 replicates these activities using a domain with 54% identity to C16. Our results reveal how vaccinia virus subverts the capacity of Ku to recognize viral DNA. Vaccinia virus expresses two proteins that interfere with the host immune response. Here, authors provide the structural basis for this viral defence mechanism, conserved in other poxviruses such as the causatives agents of smallpox and monkeypox. [ABSTRACT FROM AUTHOR]

Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1-11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane-spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O-layer inserted in the outer membrane and the I-layer inserted in the inner membrane. While the structure of the O-layer has been solved by X-ray crystallography, there is no detailed structural information on the I-layer. Using high-resolution cryo-electron microscopy and molecular modelling combined with biochemical approaches, we determined the I-layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived. [ABSTRACT FROM AUTHOR]