Increased frequency of DNA deletions in pink-eyed unstable mice carrying a mutation in the Werner syndrome gene homologue
Michel Lebel
Centre de Recherche en Cancérologie de l'Université Laval, Pavillon Hôtel-Dieu de Québec, CHUQ, 9 McMahon Street, Québec City, Québec G1R 2J6, Canada
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Abstract
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Werner syndrome (WS) is a rare autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases, including cancers. Accumulating evidence indicates that the WS gene product is involved in resolving aberrant DNA structures that may arise during the process of DNA replication and/or transcription. To estimate the frequency of DNA deletions directly in the skin of mouse embryos, mice with a deletion of part of the murine WRN helicase domain were created. These mutant mice were then crossed to the pink-eyed unstable animals, which have a 70 kb internal duplication at the pink-eyed dilution (p) gene. This report indicates that the frequency of deletion of the duplicated sequence at the p locus is elevated in mice with a mutation in the WRN allele when compared with wild-type mice. In addition, the inhibitor of topoisomerase I camptothecin also increases the frequency of deletion at the p locus. This frequency is even more elevated in WRN mutant mice treated with camptothecin. In contrast, while the inhibition of poly(ADP-ribose) polymerase (PARP) activity by 3-aminobenzamide increases the frequency of DNA deletion, mutant WRN mice are not significantly more sensitive to the inhibition of PARP activity than wild-type animals.
Abbreviations: p, pink-eyed dilution gene; PARP, poly(ADP-ribose) polymerase; WS, Werner syndrome.
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Introduction
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Werner syndrome (WS) is a rare disorder characterized by the premature onset of a number of processes associated with aging including malignancies (1,2). The gene responsible for WS (WRN) was identified by positional cloning and the gene product contains a domain homologous to the RecQ-type DNA helicases (3). The protein also possesses a 3'5' exonuclease activity in addition to its 3'5' helicase activity (48). WRN is considered a suppressor of illegitimate recombination as skin fibroblasts and lymphoblastoid cell lines made from circulating lymphocytes of WS patients exhibit variegated chromosomal translocations and deletions (911). In addition, cultured cells from heterozygotes have an intermediate sensitivity to certain DNA damaging agents compared with wild-type and homozygous mutant carriers (12). Recently, somatic cell mutation assays on peripheral red blood cells have also demonstrated a heterozygous effect in the apparent absence of DNA damage (13). Finally, it has been shown that the replication defect detected in WS lymphoblastoid cell lines is associated with an impaired S-phase transit (14). At the molecular level, the rate of initiation of DNA replication is retarded in WS cells compared with control cells (15,16).
In order to estimate the mutation rate in WS cells, most studies were carried out by manipulating cultured cells or by using peripheral blood cells (913). There is little information on the frequency of mutations in solid tissues of individuals with a mutation in the WRN gene. To estimate the frequency of DNA deletions directly in the skin of mouse embryos, without the need for cultivating cells, mice with a deletion of part of the murine WRN helicase domain were created (17). These mutant mice are referred to as WRN
hel/
hel animals. Mutant mice were then crossed to the C57BL/6Jpun/pun strain (Jackson Laboratory, Bar Harbor, ME). The C57BL/6Jpun/pun mouse strain has a 70 kb internal duplication at the pink-eyed dilution (p) gene. The p gene encodes a melanosomal integral membrane protein responsible for the assembly of a high-molecular-weight melanin complex that produces the black coat color of wild-type mice. Phenotypically, the pun mutation results in a dilute, light gray coat color. Deletion events that remove one copy of the duplication lead to a reversion of the pun mutation to wild-type p gene. One deletion event occurring in a pre-melanocyte in the embryo will cause a visible black spot on the gray fur of offspring after the amplification of the pre-melanocyte (18,19). Thus, an increase in the number of mice with black spots on their coat reflects the frequency of spontaneous DNA rearrangements. The number and the size of the black spots on each pup were recorded at 12 days of age when spots are most easily visible (18). The pigmentation of the eyes, which is also affected by the p gene status, was not analyzed in this study. As shown in Table I
, 6.2% of the WRN+/+/C57BL/6Jpun/pun pups had one or more black spots on their fur concordant with previous finding (18,19). Interestingly, 23.1% of WRN
hel/
hel/C57BL/6Jpun/pun pups had black spots on their fur. In particular, one WRN
hel/
hel/C57BL/6Jpun/pun pup had black spots over 20% of its coat. None of the WRN+/+/C57BL/6Jpun/pun pups showed such extensive spotting. In addition, this extensive spotting has not been recorded in the literature for WRN+/+/C57BL/6Jpun/pun pups (18,19). On average, each spotted animal had one or sometimes up to 10 black spots 0.10.5 cm in diameter. The range of spotting for each WRN+/+/C57BL/6Jpun/pun and WRN
hel/
hel/C57BL/6Jpun/pun pup was 2.8 and 3.1 spots per pup, respectively. However, statistical analysis (
2 test) indicated that the range of spotting per animal was not significantly different between WRN+/+/C57BL/6Jpun/pun and WRN
hel/
hel/C57BL/6Jpun/pun pups (data not shown). Although microscopic examination of the hair follicles was not performed, macroscopic observation of the animals (up to the age of 6 months) revealed that the size and the number of spots found on each mouse did not change with age. Interestingly, 11.6% of pups that were heterozygous for the WRN
hel allele also had black spots on their fur. Thus, there is also an increase in the frequency of DNA rearrangements in WRN heterozygous carriers. Although the frequency of mutation in heterozygotes is less than in homozygotes, these results strongly suggest a deleterious phenotype associated with the heterozygotes that can be of potential health concern as it has been suggested by earlier studies on WS carriers (12,13).
The frequency of black spots on the fur of C57BL/6Jpun/un offspring is elevated following exposure of the pregnant females to several carcinogenic treatments (18,19). The mouse model described in this study therefore provided an opportunity to test the effect of DNA damaging drugs on the stability of the p locus in vivo. Because WRN
hel/
hel embryonic stem cells are more sensitive to topoisomerase inhibitors than wild-type cells, pregnant females were injected with the topoisomerase I inhibitor camptothecin to examine the effect of this drug on DNA deletion directly in tissues of the embryos. In contrast, WRN
hel/
hel embryonic stem cells are not more sensitive than wild-type cells to either ionizing radiation, DNA cross linking agents or alkylating agents (17). Therefore, such treatments were not performed on these mice in this study. Topoisomerase I inhibitors such as camptothecin are known to cross the placental barrier and affect the fetus (20,21). Consequently, intraperitoneal injections were performed on females at their tenth day of gestation (10.5 days post-conception based on the presence of vaginal plug). First, a dose of 40 mg of camptothecin/kg body wt was injected into pregnant females. While WRN+/+/C57BL/6Jpun/pun females survived the treatment, all WRN
hel/
hel/C57BL/6Jpun/pun females showed extensive diarrhea and died 5 days after the intraperitoneal injection. A dose of 20 mg of camptothecin/kg body wt was thus injected into pregnant females (Table II
). Such a dose did not kill the WRN
hel/
hel/C57BL/6Jpun/pun females. Under these conditions, 68% of the WRN
hel/
hel/C57BL/6Jpun/pun offspring had black spots on their fur. In contrast, only 50% of the WRN+/+/C57BL/6Jpun/pun pups had black spots on their coat. One WRN
hel/
hel/C57BL/6Jpun/pun pup had black spots over 20% of its coat (Figure 1
) similar to the one untreated WRN
hel/
hel/C57BL/6Jpun/pun animal described earlier (Table I
). Altogether, these results indicate that camptothecin increases the frequency of deletions at the p gene in offspring, and this frequency is more elevated in mice with a mutation in both WRN alleles.
Camptothecin inhibits topoisomerase I at the DNA replication fork and causes double strand breaks. The poly(ADP-ribose) polymerase (PARP) enzyme and the Ku complex, which are involved in DNA repair, are molecules that can bind to such DNA damage (2224). The Ku complex is known to increase the activity of the WRN exonuclease domain in vitro (23,24). However, nothing is known about the effect of PARP on WRN activity. The PARP enzyme is associated with the DNA replication complex and it can regulate the activity of topoisomerase I through ribosylation (25). The WRN protein also binds to topoisomerase I in vitro (26). It has been suggested that WRN proteins act in concert with topoisomerase I as a part of a replication structure (26). As topoisomerase I, WRN protein and PARP enzymes are all detected in the DNA replication complex, it was reasonable to examine the effect of PARP inhibition on the frequency of DNA deletion in this WRN
hel/
hel/C57BL/6Jpun/pun mouse model. Thus, pregnant females were injected with 200 mg/kg of 3-aminobenzamide. Such a dosage is known to inhibit the PARP enzyme in rodent tissues (27). As shown in Table III
, 27% of WRN+/+/C57BL/6Jpun/pun and 31% of WRN
hel/
hel/C57BL/6Jpun/pun offspring had black spots on their coat. Again, one WRN
hel/
hel/C57BL/6Jpun/pun pup had black spots over 25% of its coat (Figure 1
) similar to the one untreated WRN
hel/
hel/C57BL/6Jpun/pun animal described earlier (Table I
). Whereas the inhibition of PARP activity increases the frequency of DNA deletion, the data also indicates that mutant WRN mice are not significantly more sensitive to the inhibition of PARP activity than wild-type animals. These results suggest that PARP and the WRN helicase function do not act in concert at the sites of DNA double-strand break repair as has been suggested for the Ku complex and WRN (23,24). Indeed, immunoprecipitation experiments have failed to show the co-immunoprecipitation of WRN with the PARP enzyme (data not shown).
To conclude, these experiments have demonstrated that mice with a mutation in the WRN homologue have an increased frequency of spontaneous deletion of a specific 70 kb duplicated DNA fragment at the p gene. This is the first report on the frequency of spontaneous mutation in the skin of a mouse model carrying a mutation in the WRN gene. The increased frequency of mutation in WRN mutant mice correlates with the increased incidence of tumors described in mutant mice (28). The WRN mutant mice used in this study synthesize a stable mutant WRN protein presumably with a non-functional helicase domain (17). No mutant WRN protein has been detected in any WS patients (reviewed in ref. 29). This is a major difference between this mouse model and all WS patients. Nonetheless, cells derived from this mouse model phenocopy many aspects of the human WS cells (17). This animal model clearly indicates the importance of the role of the WRN helicase domain in the suppression of deletion mutations in vivo. The absence of WRN helicase function is affecting genetic stability in melanocyte precursors during embryogenesis. It does not seem to overtly affect amplification, migration, differentiation or survival of these melanocyte precursors as 0.8% of all the WRN
hel/
hel/C57BL/6Jpun/pun mice showed extensive spotting of their coat. None of these mice showed skin lesions even during adulthood. In addition, the number and the size of black spots did not increase during adulthood indicating that deletions at the p locus do not occur frequently in mature melanocytes. Thus, the WRN helicase has an important role in maintaining the integrity of the p locus in highly proliferating pre-melanocytes of the mouse embryos.
Finally, this study clearly demonstrates the increased in vivo deleterious effects of camptothecin on animals with a mutation in the WRN gene at the genomic level. Camptothecin inhibits the re-ligation step of the topoisomerase I activity creating a topoisomeraseDNA complex or a cleavage complex. Collision of the DNA replication fork with such cleavage complexes will result in double strand breaks that can be repaired by a recombinogenic pathway. The exact recombinogenic mechanism is unknown but it is believed that the elevated intrachromosomal recombination seen in camptothecin treated cells could be due to insufficient relaxation of supercoiled DNA by topoisomerase I (30 and references therein). Under such condition, loss of a duplicated DNA fragment could be due to DNA polymerase slippage, unequal sister chromatid exchange or abnormal recombinational events. In the absence of WRN protein, the frequency of abnormal recombinational events is higher.
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Notes
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Email: michel.lebel{at}crhdq.ulaval.ca
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Acknowledgments
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I thank Dr P.Leder for the C57BL/6Jpun/pun mice and helpful discussion. I am grateful to A.Julien for technical assistance with the animals and Dr A.-M.Mes-Masson for critical reading of the manuscript. This work was supported by an operation grant from the Canadian Institutes of Health Research.
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Received August 6, 2001;
revised September 26, 2001;
accepted October 9, 2001.