Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility

Abstract

Exonuclease 1 (Exo1) is a 5'-3' exonuclease that interacts with MutS and MutL homologs and has been implicated in the excision step of DNA mismatch repair. To investigate the role of Exo1 in mammalian mismatch repair and assess its importance for tumorigenesis and meiosis, we generated an Exo1 mutant mouse line. Analysis of Exo1(-/-) cells for mismatch repair activity in vitro showed that Exo1 is required for the repair of base:base and single-base insertion/deletion mismatches in both 5' and 3' nick-directed repair. The repair defect in Exo1(-/-) cells also caused elevated microsatellite instability at a mononucleotide repeat marker and a significant increase in mutation rate at the Hprt locus. Exo1(-/-) animals displayed reduced survival and increased susceptibility to the development of lymphomas. In addition, Exo1(-/-) male and female mice were sterile because of a meiotic defect. Meiosis in Exo1(-/-) animals proceeded through prophase I; however, the chromosomes exhibited dynamic loss of chiasmata during metaphase I, resulting in meiotic failure and apoptosis. Our results show that mammalian Exo1 functions in mutation avoidance and is essential for male and female meiosis.

Figure 1

Disruption of the mouse Exo1 gene by homologous recombination. (A) Map of the targeting vector, the Exo1 wild-type locus, and the modified locus. The probe used for Southern blot analysis is indicated (red bar). (B) Southern blot analysis of HindIII-digested genomic DNA from F2 animals; +/+, wild-type; +/−, heterozygote; −/−, homozygote. (C) Northern blot analysis of poly(A) RNA from Exo1 ES cell lines using exon 6 (ex 6) and exons 7–14 (ex 7–14) probes. (D) RT–PCR analysis of total RNA from Exo1 ES cell lines using primers located in exons 5 and 7. (E) DNA sequence of the mutant RT–PCR product in Exo1^−/− mice shown in D. Note that exon 5 is spliced to exon 7 in frame, confirming the deletion of exon 6. (F) Western blot analysis of protein extracts from wild-type and Exo1^−/− ES cells using an anti-Exo1 antibody. Note the slightly faster migrating protein species in Exo1^−/− cells.

Figure 2

Mismatch repair is impaired in Exo1^−/− ES cells. DNA MMR activity was assayed in Exo1^−/−, Msh2^−/−, Exo1^−/− + Msh2^−/−, and wild-type (WT) cell extracts as described (Thomas et al. 1995). Substrates designated with Ω contain the number of extra nucleotides that accompany the symbol. Substrates are designated with a 3′ when the nick is in the (−) strand at the AvaII site (position −264), or with a 5′ when the nick is in the (−) strand at the Bsu36I site (position +276). The nucleotide position of the mismatch or unpaired bases in the lacZ complementation gene is indicated after the @, where position +1 is the first transcribed base of the lacZα complementation gene. The (−) sign designates the strand containing the extra nucleotide(s). WT, wild-type mouse ES cells. Cell extracts were prepared as described (Thomas et al. 1995) from ES cells. The results are averages based on counting >500 plaques per variable in three independent experiments. Error bars represent the standard deviations. Blue/white ratios of plaque color (not shown) demonstrated that, when observed, MMR was directed to the nicked strand.

Figure 3

Survival of Exo1 mutant mice. The survival curve was generated using the Prism (GraphPad Prism 2.0) software package. The differences between the Exo1^−/− versus Exo1^+/+ and Exo1^+/− curves are significant (p < 0.0001 and p = 0.0012, respectively). The difference between the Exo1^+/− and Exo1^+/+ curves is not significant (p = 0.2937). For comparison, the survival of Msh2^−/− mice on similar genetic background is shown (Smits et al. 2000). Statistical analysis is according to the log rank test.

Figure 4

Testis morphology in males. (A–C) Hematoxylin and eosin staining of testis sections from Exo1^+/+ (A) and Exo1^−/− (B,C) adult males. Note the unusual spindle structures within the seminiferous tubules of Exo1^−/− males (C, asterisks). (D,E) TUNEL staining to detect apoptotic cells (brown precipitate) in Exo1^+/+ (D) and Exo1^−/− (E) adult males. (E) Note the increased level of apoptosis in the testes of Exo1^−/− males. (F) Comparison of testis size in males of different Exo1 genotypes. lc, leydig cells; m, metaphase I cells; ps, pachytene spermatocytes; sg, spermatogonia; s, spermatozoa. Bars, 50 μm.

Figure 5

Chromosome pairing and synapsis during meiosis I. (A,B) electron micrographs of silver-stained chromosome spreads from Exo1^+/+ (A) and Exo1^−/− (B) spermatocytes, showing normal pairing and synapsis at pachynema. (C,D) Correct spatial and temporal localization of Mlh1 (green, FITC signal) on meiotic chromosomes from Exo1^+/+ (C) and Exo1^−/− (D) mice stained with a dual antibody against Cor1 and Syn1 and TRITC/red secondary antibody. The centromere is marked with human CREST antisera (blue, Cy5 signal). (E–H) Air-dried chromosome preparations from Exo1^+/+ (E) and Exo1^−/− (F–H) spermatocytes, showing abnormal metaphase configurations in the absence of Exo1. Although some crossovers remain (arrowheads), the majority of chromosomes are either univalents or appear to be achiasmate bivalents (arrows).

Figure 6

Ovarian morphology and meiotic progression in Exo1^−/− females. (A–C) Gross morphology of the ovaries from Exo1^+/+ (A) and Exo1^−/− (B,C) female mice at 7 mo of age. Note the loss of oocytes and the smaller size of the ovary from Exo1^−/− females. Oviductal and uterine structures look normal in the absence of Exo1. Magnification: A–C, 16×. (D–F) Chromosome spreads from oocytes taken at day 2 pp from Exo1^−/− pups. Anti-Cor1 antibody is shown in red (TRITC), anti-Dmc1/Rad51 antibody is in green (FITC), and CREST autoimmune serum, indicating the centromeres, is in blue (CY5). Oocytes from Exo1^−/− females progress through early pachynema, as demonstrated by complete synapsis of chromosomes and residual Dmc1/Rad51 foci (D), and into early diplotene (E), as demonstrated by the single cores with large regions of chromosomes remaining synapsed and the absence of Dmc1/Rad51 foci. (F) These oocytes then enter dictyate arrest, as demonstrated by the gradual degradation of chromosomal cores, but persistence of Cor1 and the clustering of centromeres.