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  • The data from the Sur lab have

    2021-02-24

    The data from the Sur lab have showed DNA-PK is involved in metabolic gene regulation in response to insulin. DNA-PK regulates fatty Cy3 carboxylic acid (non-sulfonated) sale synthesis by modulating the protein expression of fatty acid synthase (FAS) in a feeding-dependent manner. DNA-PK induces the activation of Upstream Stimulatory Factor 1 (USF-1), which sequentially binds the −65 E-box of the FAS promoter. In addition, our recent finding suggests DNA-PK may regulate the homeostasis of cell proliferation. These explorations have revealed that DNA-PK has more important roles than originally thought, so it is necessary to re-evaluate the importance of DNA-PK.
    The structure of DNA-PK DNA-PK, a member of phosphatidylinositol-3-OH kinase (PI(3)K)-related protein family [1], is a holoenzyme consisting of a catalytic subunit (DNA-PKcs) and a heterodimer of Ku (Ku70/Ku80) proteins. The catalytic subunit of DNA-PK (DNA-PKcs) comprises of 4129 amino acids (about 469kilodalton). The DNA-PKcs gene is located at 8q11 in human. Besides catalytic domain, DNA-binding and Ku-binding domains, DNA-PKcs also contain a Leucin-rich region (LRR), FAT (FRAP (FKBP12-rapamycin-associated protein), ATM (ataxiatelangiectasia mutated), TRRAP (transactivation/transformation-domain-associated protein)) domain, C-terminal of FAT domain (FATC) and two phosphorylation clusters (PQR and ABCDE) [2], [3], [4] (Fig. 1A). Heterodimer of Ku70/Ku80 consists of 609 amino acids and 732 amino acids respectively. Their genes are separately located on chromosomes 22q13 and 2q33–34 in human [5]. Previous studies have suggested a direct role of DNA-PKcs in promoting the synapsis of broken DNA ends [6], [7], most probably by self-association of DNA-end-bound DNA-PKcs molecules. The three-dimensional (3D) structure of DNA-PK complex at 25Ǻ resolution as determined by single-particle electron microscopy has shown that the binding of Ku and DNA elicits conformational changes in the FAT and FATC domains of DNA-PKcs. Observed dimeric particles have two DNA-PKcs/Ku70/Ku80 holoenzymes interacting through the N-terminal HEAT repeats [8]. The proximity of two similar complex contacting to the DNA ends suggests that these synaptic complexes maintain broken DNA ends in proximity and provide a platform for access of the various enzymes required for end processing and ligation. A higher resolution structure (7Ǻ resolution) of DNA-PKcs determined by cryo-electron microscopy single-particle reconstruction has demonstrated that this structure is composed of density rods throughout the molecule that are indicative of helices [9]. Moreover, docking of homology models into the DNA-PKcs structure demonstrates that up to eight helical HEAT repeat motifs fit well within this density rods. Furthermore, the overall fold is clearly visible in the crystal structure of human DNA-PKcs at 6.6Ǻ resolution [10]. The numerous a-helical HEAT repeats (helix–turn–helix motifs) facilitate bending and allow the polypeptide chain to fold into a hollow circular structure. The carboxy-terminal kinase domain is located on top of this structure, and a small HEAT repeat domain that probably binds DNA is inside. The structure provides a flexible cradle to promote DNA double-strand-break (DSB) repair.
    The central role of DNA-PK in non-homologous end-joining repair DNA DSBs are considered the most cytotoxic type of DNA lesion. DNA DSBs result from endogenous events such as V(D)J recombination and the production of reactive oxygen species (ROS) during cellular metabolism, as well as from exogenous sources such as ionizing radiation and radiomimetic drugs [11], [12], [13], [14]. When left unrepaired, such lesions can result in cell death. If misrepaired, DSBs have the potential to lead to chromosomal translocations and genomic instability that may result in cell death or cancer [12], [15]. NHEJ is thought to be the primary pathway for DSB repair in human somatic cell. NHEJ is initiated by binding of the ring shaped Ku70/Ku80 heterodimer (Ku) to both ends of a DSB, followed by recruitment of DNA-dependent protein kinase catalytic subunit (DNA PKcs) to the DNA-Ku complexes. Each of the Ku proteins contributes to a central DNA-binding core [16]. The N-terminus of Ku70 contains an acidic domain that is phosphorylated in vitro by DNA-PKcs [17], although this phosphorylation may not be required for NHEJ [18]; whereas the C-terminus contains an SAP (SAF-A/B, Acinus and PIAS) domain which is a putative chromatin/DNA-binding domain [2]. The c-terminal region of Ku80 forms a long flexible arm that may be involved in protein–protein interactions [19], [20] and a conserved region at the distal C-terminus, which is required for interaction with DNA-PKcs [21], [22], [23]. Ku has strong avidity for DNA with a variety of end structures without apparent sequence specificity, including blunt, over-hanged, hair-pinned and damaged ends [24].