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  • br Introduction br Structure of the Ku

    2020-05-12


    Introduction
    Structure of the Ku heterodimer associated to DNA The atomic structure of the Ku heterodimer, alone and bound to a small DNA fragment, was recently determined (Walker et al., 2001) and has significantly improved our understanding of DNA recognition by DNA-PK holoenzyme (Fig. 3). Both subunits, Ku70 and Ku80, fold into a similar structure composed of an N-terminal α/β domain, a β-barrel and a helical C-terminus arranged into an open-ring that encircles the DNA (Fig. 3A and B). A remarkable feature of this DNA–Ku complex is that the protein contacts the DNA mostly through its sugar-phosphate backbone. This provides a structural explanation for the absence of significant sequence preference in DNA recognition by the Ku heterodimer, since nucleotide bases do not participate in the interaction. Also, atomic force microscopy (AFM) studies (Yaneva et al., 1997) had already showed that Ku can translocate internally along the DNA, a behaviour again supported by the absence of bonds with specific sequences. Given the circular nature of this structure, an unresolved question is how the threaded Ku ring dissociates from the DNA after repair. Major structural rearrangements or proteolytic degradation are just two of the ideas suggested. The Ku heterodimer could only be crystallised using a truncated Ku86 construct lacking a C-terminal ∼20 kDa region of the Ku80 subunit implicated in DNA-PKcs recruitment into the Ku–DNA complex (Gell and Jackson, 1999, Singleton et al., 1999). Recently, the structure of this region has been determined using NMR spectroscopy (Harris et al., 2004; Zhang et al., 2004) and found to contain six α-helices organised into a unique fold (Fig. 3C). The structure includes two hydrophobic pockets that have been suggested to be involved in ligand binding (Harris et al., 2004).
    DNA-PKcs under the micromolar to molar microscope In EM fields DNA-PKcs molecules most commonly appear as monomeric forms with rough dimensions of 15 nm long and 7–10 nm thick (Chiu et al., 1998, Boskovic et al., 2003) (Fig. 4A and B). These images show different shapes because molecules bind to the support film of the EM grid in several orientations. Each view corresponds to a projection in a fixed set of angles and after applying classification procedures, collections of single images corresponding to similar orientations can be averaged (Fig. 4C). These average images display higher signal to noise ratio and allow visualisation of some of the structural features of the particle. Most apparent is the resolution of two domains located at opposite sides of the molecule (Fig. 4C, arrows). One side of the protein seems to hold protein mass surrounding a lower density centre or cavity (Fig. 4C, i), while in the opposite side a single segment of protein mass appears (Fig. 4C, ii).
    Visualising DNA recognition by DNA-PK One of the key steps for the cell to succeed in the repair of DNA lesions is the rapid and highly specific detection of broken DNA at the site of damage. Consequently, much interest has focused on the study of DNA binding by DNA-PK. This interaction has been extensively characterised biochemically (Leuther et al., 1999, Hammarsten et al., 2000, Martensson and Hammarsten, 2002) and with the help of surface plasmon resonance (West et al., 1998). DNA binding activates DNA-PKcs kinase activity, which is essential for progression of the NHEJ repair pathway. In attempts to get a structural view of these DNA recognition processes, AFM has been used to visualise the Ku heterodimer, DNA-PKcs and the DNA-PK holoenzyme in association with DNA (Cary et al., 1997, Yaneva et al., 1997). In these studies both Ku and DNA-PKcs were found to bind DNA independently. In the absence of DNA-PKcs, Ku could distribute along the whole DNA whereas DNA-PKcs alone was preferentially bound at DNA termini. When both Ku and DNA-PKcs were present, the two proteins associated and co-localised at the DNA end, provided that a sufficiently long DNA fragment was present. DNA fragments below 18 bp were found to be too small to accommodate the two proteins (Yaneva et al., 1997).