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  • At this stage it is still difficult

    2021-04-29

    At this stage it is still difficult to assign specific domains to some of the regions observed in these structures, although some attempts have already been made (Brewerton et al., 2004). Clearly, higher resolution data is needed. In the future, even if an atomic structure of DNA-PKcs is determined, EM of single molecules will most likely be the only approach capable of providing 3D information on the conformational changes that accompany DNA-PKcs function, and on the multi-protein complexes generated as part of NHEJ, where DNA-PK binds many other repair molecules.
    3D Structure of DNA-bound DNA-PKcs The first 3D insight into the mode of DNA recognition by the catalytic subunit of DNA-PK have recently been obtained by the use of single particle EM analysis of DNA-PKcs bound to a blunt-ended 54 bp duplex DNA (Boskovic et al., 2003). Binding of this DNA molecule greatly stimulated the activity of DNA-PKcs in phosphorylating one of its natural substrates, the XRCC4 protein, showing that interaction with this DNA was functionally relevant. Nevertheless, the binding reaction was not completely efficient and mixtures of free and bound complexes were observed in the Mifepristone microscope. Consequently, the data obtained included images from both types of complexes which had to be somehow separated into distinct data sets to be processed independently. By using a multi-model refinement approach, Boskovic and co-workers were able to obtain ‘computationally’ purified collections of images coming from either free or DNA-bound protein. This methodology allowed the reconstruction of a DNA-bound form of DNA-PKcs (Fig. 6C and D). DNA-bound DNA-PKcs shows substantial conformational changes compared to the unbound protein. The palm domain is bent and comes into contact with the head so that a small channel is left between head and palm, which is sufficiently large to accommodate double-stranded DNA. The density for the bound DNA is not directly observed at the threshold used to represent 100% of the protein mass, but appears after modifying this value slightly, probably reflecting the averaging of a range of slightly different conformations. Indeed, the atomic structure of a double-stranded DNA could be easily docked into this reconstructed DNA (Fig. 6C; the location of the DNA has been modelled according to the data from Boskovic et al. (2003)). DNA-PKcs sits at one side of this DNA, consistent with the recognition of DNA termini by this kinase. Therefore, DNA-PKcs binds double-stranded DNA and most likely the palm domain stabilises this interaction by encircling and grabbing the DNA. Interestingly, the DNA-bound conformation seems to involve an interaction between one domain found in the head and one found in the palm region, both of them displaying significant rearrangements upon DNA binding. These structural movements are likely to play a role in the activation of DNA-PKcs's kinase activity on DNA binding. Rough measurements indicated that around 18 bp could be accommodated within the space formed between head and palm domains. Several studies have demonstrated that a minimum length of ∼15 bp of dsDNA is needed to achieve proper kinase activation (Leuther et al., 1999, Hammarsten et al., 2000, Martensson and Hammarsten, 2002). Smaller DNA fragments bind with comparable affinity but they do not actually activate DNA-PKcs. More significantly, increasing the length of the DNA fragments much above 20 bp does not further stimulate this activity. The 3D structure of the DNA–DNA-PKcs complex provides some clues to the molecular basis of this behaviour. Most likely, DNA-PKcs kinase activation can result from the interaction between domains in the head and the palm upon DNA binding. Hence, the observed conformational change might only be stabilised when some specific regions of the protein contact the sugar-phosphate backbone of the DNA and only a DNA which is sufficiently long to fill the cavity within DNA-PKcs could activate the protein. Longer DNAs would not add further stability or kinase activity. At the site of damage this DNA–DNA-PKcs complex will most likely be found as part of a larger assembly containing the Ku protein. We have hypothesised how this complex could look like by combining data from single-particle EM (Boskovic et al., 2003), the crystal structure of the Ku86:70–DNA complex (Walker et al., 2001), and the NMR structure of the C-terminal domain from Ku86 (Harris et al., 2004) (Fig. 6E; blue colour for the DNA in the complex). Nevertheless, the actual 3D structure of such a complex remains to be defined experimentally.