Generation and characterization of endonuclease G null mice

Abstract

Endonuclease G (endo G) is one of the most abundant nucleases in eukaryotic cells. It is encoded in the nucleus and imported to the mitochondrial intermembrane space. This nuclease is active on single- and double-stranded DNA. We genetically disrupted the endo G gene in mice without disturbing a conserved, overlapping gene of unknown function that is oriented tail to tail with the endo G gene. In these mice, the production of endo G protein is not detected, and the disruption abolishes the nuclease activity of endo G. The absence of endo G has no effect on mitochondrial DNA copy number, structure, or mutation rate over the first five generations. There is also no obvious effect on nuclear DNA degradation in standard apoptosis assays. The endo G null mice are viable and show no age-related or generational abnormalities anatomically or histologically. We infer that this highly conserved protein has no mitochondrial or apoptosis function that can discerned by the assays described here and that it may have a function yet to be determined. The early embryonic lethality of endo G null mice recently reported by others may be due to the disruption of the gene that overlaps the endo G gene.

FIG.1.

Disruption of the murine endo G gene and characterization of expression. A. endo G genomic locus. The murine endo G locus is shown on the top line. The blocks of homology used for exchanging exon 2 and replacing it with the neo gene are shown below the endo G locus map. The configuration of the disrupted (targeted) locus is shown in the third line. The positions of the diphtheria toxin gene (DT), untranslated region (UTR), and primer sites (black arrowheads with white numbers) are indicated. The gene that overlaps with the endo G locus is D2Wsu81e and is shown shaded; the most downstream exon of this gene overlaps with all of endo G exon 3. B. PCR assay for the wild-type (endo G^+/+ [+/+]) and genetically disrupted alleles (endo G^+/− [+/−] and endo G^−/− [−/−]). Primers 8 and 9 are used to identify the wild-type allele, and these primers generate a 530-bp product (Table 1). Primers 1 and 5 (Table 1) are used to detect the disrupted (mutant or KO) allele, and these primers generate a band of approximately 1.6 kb. The positions of molecular size standards (MWM) (in kilobases) are indicated to the left of the gels. C. Southern blot analysis of the wild-type and disrupted allele. The probe indicated in Fig. 1A was used to hybridize to genomic DNA resolved after NsiI digestion. The wild-type allele yields a band of 9 kb, and the disrupted allele yields a band of 5.9 kb. D. Western blot analysis of endo G production in wild-type and null mice. A polyclonal antibody was used to develop a Western blot to reveal the 28-kDa mature form of endo G. No immunoreactive band was detected down to approximately 6 kDa on this and other replicate Western blots.

FIG. 2.

Recombinant forms of the wild-type and mutant forms of the endo G protein and nuclease activity assays. A. Recombinant forms of the myc epitope-tagged mature endo G and the predicted truncated form (120 aa from exon 1 plus 3 aa from exon 3) of endo G with a 21-aa his-myc C-terminal epitope tag (used for purification). Expression vectors indicated at the top of each lane were transfected into 293 cells, and the proteins were purified with anti-myc immunobeads (see Materials and Methods). The full-length (FL) recombinant mature form migrates at the expected position of 30 kDa (250 aa for the mature form plus 21 aa for the his-myc tag), as seen on this Western blot (arrow). The truncated (ΔΕ2 for exon 2 deleted) recombinant form is expected to migrate at 21 kDa (167 aa plus the 21-aa his-myc tag) before processing and 16 kDa (123 aa plus the 21-aa his-myc tag) after processing (arrow). The truncated processed form (16 kDa) migrates at a slightly slower position than expected (∼19 kDa). The immunoglobulin light chain (IgL) band is indicated. There is a cross-reactive band above the IgL band in the ΔE2 lane, which may represent a proteolytic fragment of the c-myc protein. The cross-reactive band in the FL lane at 35 kDa is a cross-reactive band of unknown identity. B. Substrate for assay of nuclease activity of the wild-type and truncated forms of endo G. R-loops were generated using T7 RNA polymerase transcription through a murine immunoglobulin class switch region as described previously. The switch region is located between positions 1 and 656 bp on the plasmid, pTW-SS22. C. Nuclease assay of the wild-type and truncated forms of endo G. The recombinant full-length (FL) endo G or the truncated form of the protein were incubated with the T7 RNA polymerase-transcribed pTW-SS22 plasmid (R-loop conformation of the substrate) (left gel) or with nontranscribed pTW-SS22 (right gel). The pTW-SS22 plasmid was linearized at XmnI. Breaks due to endo G anywhere within the R-loop switch region generated a distribution of linear products between 1,726 and 2,382 bp and another distribution between 850 and 1,506 bp, as seen in the FL lane of the left gel. Overexposure of this gel showed absolutely no evidence of R-loop cleavage activity in the ΔE2 lane of the left gel, and the intact full-length linearized plasmid band in the ΔE2 lane indicates that endo G has no nicking or dsDNA nuclease activity on duplex DNA. In the right gel, full-length recombinant endo G does have a small amount of nicking activity (relative to activity on the R-loop) on duplex supercoiled DNA, as described previously (7, 8, 10, 11, 17).

FIG. 3.

Mitochondrial genome copy number and gross structure. A. Diagram of the murine mitochondrial genome. The D-loop region of the mitochondrial genome is shown along with restriction sites. B. Southern blots of the murine mitochondrial genome. Fragments (see Materials and Methods) from the murine mitochondrial genome were used as probes on restricted mtDNA harvested from equivalent amounts of liver DNA from wild-type (+/+) and two endo G^−/− (−/−) mice. No differences in intensity, sizes of specific bands, or variations in the distribution of DNA (smearing) around each band were noted as this or darker exposures.

FIG. 4.

Apoptosis assays on cells from wild-type (+/+) or endo G^−/− (−/−) mice. A. DNA laddering in DNA from untreated (U) cells or cells treated with etoposide (E) or actinomycin D (A). Isolated splenocytes were resuspended in RPMI 1640 containing 10% FBS and 50 μM 2-mercaptoethanol with no additional treatment (U) or with 50 μM etoposide (E) or 500 nM actinomycin D (D) to a density of 0.5 × 10⁶ cells/ml. For each treatment, 12 ml of cells (6 × 10⁶ cells) were transferred to a single 10-cm-diameter dish and incubated for 12 h at 37°C and 5% CO₂. Genomic DNA was purified from 5 × 10⁶ cells, dissolved in 30 μl of TE, and resolved on a 1.5% LE agarose gel. The positions of molecular size standards (MWM) (in kilobases) are indicated to the left of the gels. B. Nuclear fragmentation assay. Cells (10⁶ cells) from the same dishes were fixed in 3.7% formaldehyde and stained with DAPI (see Materials and Methods). A minimum of 200 nuclei were analyzed for chromatin condensation by fluorescence microscopy per treatment arm of the experiment. C. Quantitation of nuclear fragmentation assay. Histograms of the data derived from panel B.