(A) Schematic diagram of wild type and mutant Atad5 loci. Exons 17–19 are indicated as solid boxes. LacZ and NEO indicate the location of the cassette that disrupted Atad5 gene expression by insertional mutagenesis. Arrows and numbers between arrows indicate the locations of primers used for genotyping and expected sizes of PCR products, respectively. PCR with gray and black primer that can bind wild type locus generates 542 bp PCR products from wild type loci but not in mutant locus due to the inserted LacZ-NEO cassette. In contrast, PCR with gray and another black primer that can bind LacZ sequence generates 900 bp PCR product only from mutant locus. (B) PCR based genotyping of DNAs isolated from mouse embryos at day 3.5 p.c. Wild type and mutant alleles of Atad5 are represented by + and m, respectively. 1^st and 2^nd lanes in each sample shows PCR products generated from wild type locus (542 bp) and mutant locus (900 bp) explained in the legend A. (C) RT-PCR analysis of Atad5 mRNA levels in wild type and Atad5^+/m MEFs. The exons to which each Atad5 primers bind are indicated on the x-axis. (D) Western blot analysis of Atad5 in wild type MEFs and Atad5^+/m MEFs. (E) β galactosidase expression in wild type (Atad5^+/+; negative control), Atad5^+/m MEF cells, and HEK 293T cells transiently transfected with a plasmid expressing β-galactosidase (positive control) were examined by immunostaining with a β-galactosidase antibody. (F) The fused Atad5-LacZ transcript was confirmed by RT-PCR. M stands for molecular marker. A 210 bp RT-PCR product only from Atad5^+/m cDNA MEF cells was detected. (G) β galactosidase expression in wild type (Atad5^+/+; negative control), Atad5^+/m MEF cells, and HEK 293T cells transiently transfected with a plasmid expressing β-galactosidase (positive control) were determined by Western blot analysis. (H) Atad5- β-galactosidase fusion protein was not detected in the Atad5^+/m MEF cells even after 12 hours recovery from 0.01% MMS treatment for one hour by immunostaining with a β-galactosidase antibody. Top and bottom panels are images of β-galactosidase antibody staining only and merged with DAPI, respectively.

(A) Atad5^+/m mice exhibited an increase in IR-induced MN-RETs. Wild type (Atad5^+/+) and SCID mice were treated with IR and their peripheral blood cells were analyzed in the same manner for comparison. CD71 and propidium iodide (PI) were used to detect RET cells and micronuclei, respectively. Fold induction after IR irradiation in each mouse with a different genotype normalized to MN-RET from unirradiated mice is presented in the graph. Five wild type, five Atad5^+/m, and two SCID mice were analyzed. (B) MEFs derived from the Atad5^+/m mice produced more chromatid breaks than wild type MEFs in response to MMS treatment. Percentages of metaphases having noted number of breaks are presented as graphs. Noticeable chromatid breaks per cell were quantified for 50 metaphases from both wild type and the Atad5^+/m MEFs. (C) MEFs derived from the Atad5^+/m mice have a high level of aneuploidy. (D) Survival of wild type and the Atad5^+/m MEFs following MMS treatment is displayed as the percentage of viable cells relative to untreated cells. P-values were calculated or each data point as follows, 0.00125% MMS (p = 0.05); 0.0025% MMS (p = 0.002), 0.005% MMS (p = 0.005), 0.0075% MMS (p = 0.09); and 0.01% MMS (p = 0.03), respectively.

(A) Atad5^+/m MEFs are defective in DNA repair. Broken DNAs after 0.01% MMS treatment were visualized by pulse field gel electrophoresis. (B, C) Atad5^+/m MEFs display high levels of spontaneous Rad51 foci in the nucleus. B and C show, respectively, an actual image and a graphic presentation of the number of Rad51 foci in the nucleus of each cell type. (D) Atad5^+/m MEFs display similar rates of sister chromatid exchange rate compared to wild type MEFs. (E, F) Atad5^+/m MEFs display high level of γH2AX foci in the nucleus. An actual image (E) and a graphic presentation (F) of the number of cells having different types of γH2AX foci in the nucleus from each cell type are shown. (G, H, I, J) Atad5^+/m MEFs undergo apoptosis at the 25^th passage. FACS analysis of wild type (G, H) and Atad5^+/m MEFs (I, J) from the 4^th passage (G, I) and the 25^th passage (H, J) were performed based on their DNA contents. Bars in images for Atad5^+/+ and Atad5^+/m (B and E) indicate 20 µm and 10 µm, respectively.

(A) Kaplan-Meier survival graph of wild type (Atad5^+/+; n = 20) and the Atad5^+/m mice (n = 22). (B) Representative H&E stained sections of tumors that arose in the Atad5^+/m mice; (I) Harderian gland adenoma, (II) hemangioma, (III) lung bronchiolar adenoma, (IV) thymoma, and (V) sarcoma from bone marrow. The panel on the right is a two-fold magnification of panel on the left. (C) Frequency of genomic copy number gains and losses among four tumors from the Atad5^+/m mice, plotted as a function of genome location with all chromosomes starting from 1pter (left) to X (right). Vertical lines indicate chromosome boundaries. Positive and negative values indicate the number of tumors that showed copy number gains (+) and losses (−), respectively. (D) The pathway activated in two tumors from the Atad5^+/m mice analyzed by microarray. Red and green colors represent the activation and inactivation in tumors, respectively.

(A) Schematic representation of the ATAD5 protein showing positions of somatic mutations (arrows) relative to the AAA-ATPase domain. Truncating (boxed), missense (black) and synonymous (gray) mutations are distinguished. Amino acids are numbered. (B) The ATAD5-R1414X mutant is associated with reduced protein levels. HEK 293T cells were transfected with constructs expressing wild type (WT) ATAD5 or mutant ATAD5. Soluble fractions, which include cytoplasmic and nucleoplasmic proteins, and chromatin-bound fractions were separated by SDS-PAGE after 48 h. Relative expression of wild type or mutant ATAD5 was determined by Western blotting with anti-FLAG-antibody. Histone H3 and tubulin were used as loading controls for chromatin-bound and soluble fractions respectively. Total RNAs, extracted from cells used in the Western blot analysis, served as templates for RT-PCR. ATAD5 mRNA expression levels determined by quantitative RT-PCR, were normalized to β-actin expression levels and are represented as a relative value compared to that of vector transfectants. (C) The ATAD5-E723X mutant protein does not interact with RFC4. Chromatin-bound fractions from HEK 293T cells transfected with constructs expressing wild type or mutant ATAD5 were immunoprecipitated (IP) with anti-RFC4 antibody or IgG control antibody, and subjected to Western blot analysis.