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  • E7046 More recently Boskovic et al have visually corroborate

    2019-08-28

    More recently, Boskovic et al. (2003) have visually corroborated that purified DNA-PKcs can bind short DNA fragments by using single particle EM. DNA biotinylated at the 5′ end was labelled by coupling to commercial streptavidin–gold particles (Fig. 4D). When analysed in the microscope, DNA-PKcs molecules with a gold density (Fig. 4E and F) were clearly visible, indicating that they were bound to the labelled DNA. DeFazio et al. (2002) have used a brilliant combination of biochemical and EM experiments to visualise a synaptic complex. Synapsis takes place where the two broken DNA ends are brought together to facilitate ligation of their backbones, to restore genomic integrity. Several repair factors must contribute to this synaptic complex and DeFazio and co-workers have used ‘pull-down’ assays to detect a DNA-PKcs-mediated synaptic activity. The putative synaptic complexes were then prepared for observation under the transmission E7046 microscope (Fig. 5). When DNA-PKcs was incubated with linear DNA, a large fraction of the DNA molecules could be observed containing a DNA-PKcs molecule bound to their ends. More significantly, a substantial proportion of these DNA molecules formed circular structures in which the two ends were bridged by interacting pairs of DNA-PKcs molecules. When both Ku and DNA-PKcs were present in the reactions, there was a 4-fold increase in the amount of DNA molecules with two ligated ends. All these synaptic complexes formed in the presence of the kinase inhibitor wortmannin revealing that synapsis is independent of the kinase activity of DNA-PKcs. In conclusion, DeFazio and co-workers visually demonstrate that DNA-PKcs is capable of maintaining two DNA ends together in a configuration compatible with a subsequent ligation. Thus, DNA-PKcs is probably the best candidate for an end-to-end synaptic factor.
    3D Structures of DNA-PK catalytic subunit (DNA-PKcs) using electron microscopy A deep understanding of the structural basis of DNA-PKcs function will only come when an atomic structure of this large protein is determined. In the meantime, EM has already provided three structures of DNA-PKcs at medium resolution. Despite all of these three structures having been obtained using EM, each of them has applied a different methodology and their comparison is not entirely straightforward. Although negatively-stained samples were used in two studies (Leuther et al., 1999, Boskovic et al., 2003) one used two-dimensional crystals of the protein while the other utilised isolated molecules (Boskovic et al., 2003). A third study has also used single-particle analysis though applied to images of unstained protein obtained under liquid nitrogen temperatures (cryoEM) (Chiu et al., 1998). The two stained structures are quite compatible despite the differences in the conditions used to prepare the sample. Both structures show that DNA-PKcs is divided into three large regions, labelled as ‘head’, ‘palm’, and an ‘arm’ connecting head and palm (Fig. 6A and B). The head encloses a cavity surrounded by protein density and leaves several openings. Most differences between the two stained structures lie at the level of the palm which in the single particle reconstruction seems to be rotated compared to the crystal structure. Since the crystal packing involves lateral interactions between the head of one DNA-PKcs molecule and the palm of another (Leuther et al., 1999), these contacts might account for the different conformation found in this region. Consequently, it could be reasoned that the conformation found in isolated molecules (Boskovic et al., 2003) should be closer to the native functional state. On the other hand, the 3D structure obtained from vitrified single particles shows a general architecture roughly compatible with the other two volumes, but surprisingly the structure contains many holes and cavities (Chiu et al., 1998). New cryoEM experiments will be probably required to solve these ambiguities.