Abstract

Werner syndrome is an autosomal recessive disease characterized by many age-associated disorders. At puberty, Werner Syndrome patients develop phenotypes associated with aging, including: graying of hair, type II diabetes mellitus, atherosclerosis and osteoporosis. Werner syndrome results from mutations in the REQL2 gene that result in truncated proteins lacking the nuclear localization signal.

Multiple studies have indicated a function of the Werner protein in telomere processing, but the nature of this function remains unclear. Primary fibroblasts explanted from Werner Syndrome patients are hypersensitive to a variety of genotoxic agents that generate double-strand breaks in dividing and nondividing cells, or generate stalled replication forks in dividing cells. In this work, we demonstrate that fibroblasts derived from Werner Syndrome patients are defective in both the nonhomologous end joining pathway and in homologous recombination:

Nondividing Werner syndrome fibroblasts demonstrate an increased sensitivity to induction of growth arrest in response to oxidative stress associated with accumulation of telomere dysfunction induced foci. That this sensitivity is due to dysfunction or absence of the Werner protein was demonstrated by stable expression of dominant-negative Werner protein or by the use of RNAi to inhibit Werner protein synthesis in normal fibroblasts. Werner deficient normal human diploid fibroblasts displayed sensitivity to growth arrest and induction of TIFs. The growth arrest and telomere dysfunction induced foci were suppressed in Werner syndrome fibroblasts with stable expression of human telomerase reverse transcriptase transgene, a telomere-specific enzyme that elongates telomeres. Further, by reversibly expressing telomerase on a retroviral insertion vector containing flanking loxP sites, we observed that long-telomere Werner syndrome fibroblasts were resistant to the growth arrest induced by oxidative stress. This indicates that the suppression is attributable to long telomeres, not telomerase expression.

In the absence of WRN, rapid telomere erosion signals a growth arrest. Consistent with these observations, we found that p53-inhibited WS fibroblasts containing TIFs undergo extensive telomere deletions, indicating that these lesions are not repaired without Werner. Further, we provide direct evidence that Werner undergoes nuclear translocation into aggregates coincident with telomere dysfunction induced foci in nondividing normal human diploid fibroblasts treated with hydrogen peroxide. In conclusion, these data suggest that the Werner Syndrome protein functions in double-strand break repair at telomeres in nondividing normal human diploid fibroblasts. The data further indicate that additional processes, aside from the end replication problem, may contribute to replicative senescence in nondividing cells.

Because the increased frequency of Telomere Sister Chromatid Exchange and Sister Telomere Loss observed in WS fibroblasts after a pulse of H ₂O₂ is more likely a result of HR rather than NHEJ, and because we observe that agents that stall replication fork progression increase the frequency of both events, we investigated the putative role of WRN protein in DSBR of telomere DNA in proliferating cells via the HR pathway. We observe that aphidicolin-induced replication fork arrest in WS cells results in extensive Sister Telomere Loss. We also observe a 90% increase in the frequency of Telomere Sister Chromatid Exchanges. Human Papilloma Virus E6-forced replication of Werner syndrome fibroblasts after treatment with aphidicolin or hydrogen peroxide revealed the effect of stalled replication forks in the absence of WRN protein at telomeres. We observe that 50% of all telomere repeats are lost per cellular division, confirming a role of WRN protein in repair of double strand breaks within replication forks.

In conclusion, our data suggest a role of the WRN protein in repair of telomere damage in both dividing and nondividing cells. In the absence of Werner protein, inefficient replication and repair of telomere repeats sequences results in short or dysfunctional telomeres that signal a p53-dependent premature growth arrest.