Fig 2: Phylogenetics and insertion sites of KU70 derived retrocopies (A) Once the five NUKU retrocopies had been identified in the human genome, orthologous retrocopies were identified in other primate genome projects through inspection of the syntenic target sites. A tree of these sequences is shown. Unless indicated, none of the genes on the tree contain introns. Bootstrap values generated with the maximum likelihood method are shown. Marmoset NUKU3 was verified to be orthologous to the other NUKU3 sequences by target site analysis. NUKU2 and NUKU4 are apparent in the marmoset genome, but are almost completely deleted, and therefore they were not included in the alignment used to make the tree. We were unable to locate the syntenic region of NUKU1 in the Marmoset genome, indicating that this region may have been deleted (Supplementary Figure S2). Marmoset-specific retrocopies were not named but are designated by the chromosome on which they are found. *target site of NUKU5 and NUKU6 insertion is empty in Old World monkeys. (B) Based on the phylogenetic analysis and target site inspection, NUKU1–NUKU4 predate the split between Old World monkeys, New World monkeys, and hominoids. NUKU5 is specific to the great ape genomes analyzed and NUKU6 appears to be unique to the snub-nosed monkey. The time of acquisition of NUKU6 is somewhat uncertain due to the low bootstrap support in the phylogenetic analysis. The marmoset genome has birthed 9 additional KU70-like retrocopies.

Fig 3: Detection of a putative KU70 retrocopy-encoded protein (A) A heat map representation of Ribo-seq data reveals probable translation of several NUKU retrocopies. Many of the short (~30bp) Ribo-seq reads identically match more than one gene and are classified as “not unique.” The remaining reads align uniquely to either KU70 or one retrocopy. The mean proportion of reads aligning to each gene in three tissues is shown (n = 3). (B) Identification of an anti-Ku70 antibody that recognizes Nuku2p, Nuku5p, and Ku70p. The ORFs of KU70, NUKU2, and NUKU5 were fused to the GAL4 AD and expressed in yeast. A Western blot of these proteins shows that a single polyclonal anti-Ku70 antibody recognizes all three Nuku fusion proteins (three independent transformants of Nuku5-AD are shown). (C) Whole-cell protein lysates from testis, brain, and HEK293T cells were purchased or cultured. The anti-Ku70 antibody characterized in panel A was used to probe these extracts.

Fig 4: AR anchors the Ku70 and Ku80 DNA repair proteins to RNA Pol II and prevents RNA Pol II–associated DNA damage. (A) Co-IP analysis with anti-AR and anti-RNA pol II antibodies in WM1366 melanoma cells and immunoblotted for indicted proteins. (B) PLAs of WM1366 melanoma cells with antibodies against AR and Ku70 and nonimmune IgGs as specificity control. Red fluorescence puncta resulting from the juxtaposition of anti-AR and anti-Ku70/Ku80 antibodies were visualized by confocal microscope with concomitant DAPI nuclear staining. Shown are representative images and quantification of the number of puncta per cell. For this and following panels, n (cells per condition) > 50; ***, P < 0.005; ****, P < 0.001, 2-tailed unpaired t test. (C) PLAs of AR and Ku70 association in melanoma cell lines with elevated (WM1366 and SKMEL28) versus low (SKMEL5) levels of total AR protein (as shown in Fig. 1 E), with or without shRNA-mediated AR gene silencing. ***, P < 0.005; ****, P < 0.001. (D) PLAs of AR and Ku70 association in the same melanoma cell lines as in C with or without treatment with the AR inhibitor AZD3514 (10 µM for 48 h). ***, P < 0.005; ****, P < 0.001. (E and F) PLAs of the indicated melanoma cell lines with or without AR gene silencing with antibodies against Ku70 or Ku80 and total RNA Pol II (E) or elongating form (CTD Ser2 phosphorylated; F). ****, P < 0.001. (G) PLAs of melanoma cells with or without AR gene silencing as in F, with antibodies against the elongating form of RNA Pol II and ?-H2AX. Shown are better representative images. Scale bars: 10 µm. ****, P < 0.001. (H) Diagrammatic model of the AR anchoring function, required for Ku70/Ku80 association to the RNA Pol II transcription complex and prevention of transcription-associated DNA damage.

Fig 5: The primary cilium maintains the ETO-induced DNA damage response.Inhibition of ciliogenesis decreased ETO-induced DNA-PK activation. A Depletion of CEP164 inhibited ETO-induced DNA-PK activation. Extracts of RPE1 cells transfected with siRNA against CEP164 in the presence of ETO were analyzed by immunoblotting with antibodies against CEP164 and tubulin (Tub.). B Quantitative results of the frequency of ciliated CEP164-deficient RPE1 cells in the presence of ETO. The results are presented as the mean ± SD of three independent experiments; more than 100 cells were counted in each individual group. C Extracts of RPE1 cells transfected with siRNA against CEP164 were analyzed by immunoblotting with antibodies against phosphorylated DNA-PKcs (p-PKcs), DNA-PKcs (PKcs), and tubulin (Tub.). D Depletion of CEP164 inhibited ETO-induced p53 activation. Extracts of RPE1 cells transfected with siRNA against CEP164 in the presence of different concentrations of ETO were analyzed by immunoblotting with antibodies against p53 and Ku70. Removal of ETO reduced ETO-induced ciliogenesis in a time dependent manner. E Schematic representation of the experimental procedure used for F. Cells were treated with ETO for 24 h followed by culturing in ETO-free medium for additional 6 or 24 h before harvesting. F Quantitative results of the frequency of ETO-treated ciliated RPE1 cells after ETO washout. The results are presented as the mean ± SD of three independent experiments; more than 100 cells were counted in each individual group. **P < 0.01; ***P < 0.001.