A deletion within the murine Werner syndrome helicase induces sensitivity to inhibitors of topoisomerase and loss of cellular proliferative capacity

Abstract

Werner syndrome (WS) is an autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases. The gene responsible for WS encodes a member of the RecQ-like subfamily of DNA helicases. Here we show that its murine homologue maps to murine chromosome 8 in a region syntenic with the human WRN gene. We have deleted a segment of this gene and created Wrn-deficient embryonic stem (ES) cells and WS mice. While displaying reduced embryonic survival, live-born WS mice otherwise appear normal during their first year of life. Nonetheless, although several DNA repair systems are apparently intact in homozygous WS ES cells, such cells display a higher mutation rate and are significantly more sensitive to topoisomerase inhibitors (especially camptothecin) than are wild-type ES cells. Furthermore, mouse embryo fibroblasts derived from homozygous WS embryos show premature loss of proliferative capacity. At the molecular level, wild-type, but not mutant, WS protein copurifies through a series of centrifugation and chromatography steps with a multiprotein DNA replication complex.

Figure 1

Mapping of the murine Wrn gene. (Upper) Maps from the Jackson BSB and BSS backcrosses showing part of chromosome 8. Maps are depicted with centromere toward the top. A 3-centimorgan scale is shown at right. Loci mappings to the same position are listed alphabetically. Missing typing was inferred from surrounding data, where assignment was unambiguous. (Lower) Haplotype figure from the Jackson BSS backcross showing part of chromosome 8 with loci linked to Wrn. Loci are listed in order with the most proximal at the top. Solid boxes represent the C57BL6/JEi allele and open boxes the SPRET/Ei allele. The number of the animals with each haplotype is given at the bottom of each column of boxes. The percent recombination (R) between adjacent loci is given to the right with the standard error. Missing typing was inferred from surrounding data, where assignment was unambiguous.

Figure 2

Targeted disruption of the helicase domain of the murine Wrn gene. (A) Schematic representations of the genomic locus surrounding the targeted exons, the targeting vector, and the targeted locus. Open boxes represent exons coding for motifs III, IV, V, and VI of the helicase domain. Transcriptional directions of the neomycin (neo) and the thymidine (tk) genes are indicated by arrows. Before electroporation, the targeting vector was linearized at the NotI site of the vector (not shown). The length of the EcoRV diagnostic restriction fragments hybridizing to the external probe are indicated by the double-headed arrows. B, BamHI; E, EcoRI; RV, EcoRV. (B) Southern blot analysis of the tail DNA from several wt, heterozygous, and homozygous mice and two homozygous ES clones. Genomic DNA was digested with EcoRV and hybridized to the probe indicated in A. The wt band is ≈16 kb and the disrupted allele (mt) generates a 9.5 kb band. (C) Northern blot analysis of the mRNA purified from the kidneys of wt, heterozygous, and homozygous mice. Five micrograms of poly(A)⁺ RNA was loaded in each lane. The membrane was hybridized with a labeled 3.1-kb cDNA fragment of Wrn. Arrows indicate the wt 6.6-kb full-length (wt) and mutant (mt) transcripts. (D) RT-PCR analysis of purified total RNA from the spleen of wt, heterozygous, and homozygous mice. cDNA was synthesized and used as template for amplification with the primers as described. The primer set directs the amplification of 1,116- and 753-bp fragments from the wt and mutant (mt) transcripts, respectively. PCR products were blotted and the membrane was hybridized with an internal oligonucleotide. (E) Western blot analysis of WS protein expression in wt, heterozygous, and homozygous ES clones. Arrows on left indicate the wt and the mutant (mt) proteins detected with the anti-WS helicase rabbit antibody. Arrows on right indicates the approximate molecular mass of the wt (170 kDa) and mutant (156 kDa) proteins.

Figure 3

Mutagenicity and differential effects of DNA damaging agents on wt and WS ES cells. Approximately 5,000 ES cells were plated onto a feeder layer and plates were treated for 2 hr with the indicated chemicals or irradiated, and then cells were grown for 7 days to form individual colonies. Each curve represents two independent clones plated in duplicate. Graphs represent the clonogenic survival of wt Wrn^+/+, heterozygous Wrn^+/−, and homozygous Wrn^−/− ES cells after treatment with increasing concentrations of 6-thioguanine (A), type II topoisomerase inhibitor etoposide (B), topoisomerase I inhibitor camptothecin (C), increasing doses of γ-rays (D), increasing doses of UV light (E), and increasing concentration of mitomycin C (F).

Figure 4

Differential saturation density and growth properties of wt and WS MEFs. Saturation density and growth analysis of MEFs at passage 5. Cells (5 × 10⁴) from Wrn^+/+, Wrn^+/−, and Wrn^−/− were plated in 6-well plates as described. Cell number at each time point represents the average of two independent cell lines.

Figure 5

Copurification of WS protein and the multiprotein DNA replication complex is disrupted by the Wrn mutation. (A) Total cell lysates (50 μg) from wt (+/+) (lane 1) and homozygous (−/−) ES cells (lane 2) were loaded on the gel as reference. Lanes 3 and 4 contain protein from heterozygous (+/−) ES cells fractionated on a sucrose cushion as described. Lane 3, protein from the HS-4 supernatant fraction; lane 4, protein from the HSP-4 interphase fraction containing protein complexes. Proteins were size separated on 10% SDS/PAGE and detected by Western blot analysis with the anti-WS antibody. Arrows indicate the wt and the mutant (mt) WS proteins. Lanes 3 and 4 from the same autoradiogram were exposed for longer than lanes 1 and 2. (B) Immunoblot analysis for the presence of WS protein in DE52 low and high salt eluates. Lanes: 1, total cell lysate from wt ES cells; 2, protein (20 μg) from the high salt DE52 eluate; 3, protein (20 μg) from the DE52 wash fraction. Arrow indicates the wt protein.