Mre11 modulates the fidelity of fusion between short telomeres in human cells.

The loss of telomere function can result in the fusion of telomeres with other telomeric loci, or non-telomeric double-stranded DNA breaks. Sequence analysis of fusion events between short dysfunctional telomeres in human cells has revealed that fusion is characterized by a distinct molecular signature consisting of extensive deletions and micro-homology at the fusion points. This signature is consistent with alternative error-prone end-joining processes. We have examined the role that Mre11 may play in the fusion of short telomeres in human cells; to do this, we have analysed telomere fusion events in cells derived from ataxia-telangiectasia-like disorder (ATLD) patients that exhibit hypomorphic mutations in MRE11. The telomere dynamics of ATLD fibroblasts were indistinguishable from wild-type fibroblasts and they were proficient in the fusion of short telomeres. However, we observed a high frequency of insertion of DNA sequences at the fusion points that created localized sequence duplications. These data indicate that Mre11 plays a role in the fusion of short dysfunctional telomeres in human cells and are consistent with the hypothesis that as part of the MRN complex it serves to stabilize the joining complex, thereby controlling the fidelity of the fusion reaction.

Telomere dynamics during replicative senescence are not directly modulated by conditions of oxidative stress in IMR90 fibroblast cells.

The replicative lifespan of many cell types is determined by the length of telomeres in the initiating cell population. In 20% oxygen, IMR90 cells have a shorter replicative lifespan compared to that achieved in conditions that lower the levels of oxidative stress. We sought to address the role of telomere dynamics in determining the replicative lifespan of IMR90 cells. We analysed clonal populations cultured in parallel in 3 and 20% oxygen. We observed that, at senescence, telomere length was shorter in 3% oxygen and this was proportional to the lifespan extension. We observed no detectable difference in the rate of telomere erosion in the two culture conditions, however as the cells approached senescence the growth rate of the cultures slowed with a commensurate increase in the rate of telomere erosion. We conclude that, in 20% oxygen senescence of IMR90 is telomere-independent, but telomere-dependent in 3% oxygen.

Short telomeres are preferentially elongated by telomerase in human cells.

Short telomeres have been shown to be preferentially elongated in both yeast and mouse models. We examined this in human cells, by utilising cells with large allelic telomere length differentials and observing the relative rates of elongation following the expression of hTERT. We observed that short telomeres are gradually elongated in the first 26 PDs of growth, whereas the longer telomeres displayed limited elongation in this period. Telomeres coalesced at similar lengths irrespective of their length prior to the expression of hTERT. These data indicate that short telomeres are marked for gradual elongation to a cell strain specific length threshold.

The nature of telomere fusion and a definition of the critical telomere length in human cells.

The loss of telomere function can result in telomeric fusion events that lead to the types of genomic rearrangements, such as nonreciprocal translocations, that typify early-stage carcinogenesis. By using single-molecule approaches to characterize fusion events, we provide a functional definition of fusogenic telomeres in human cells. We show that approximately half of the fusion events contained no canonical telomere repeats at the fusion point; of those that did, the longest was 12.8 repeats. Furthermore, in addition to end-replication losses, human telomeres are subjected to large-scale deletion events that occur in the presence or absence of telomerase. Here we show that these telomeres are fusogenic, and thus despite the majority of telomeres being maintained at a stable length in normal human cells, a subset of stochastically shortened telomeres can potentially cause chromosomal instability. Telomere fusion was accompanied by the deletion of one or both telomeres extending several kilobases into the telomere-adjacent DNA, and microhomology was observed at the fusion points. This contrasted with telomere fusion that was observed following the experimental disruption of TRF2. The distinct error-prone mutational profile of fusion between critically shortened telomeres in human cells was reminiscent of Ku-independent microhomology-mediated end-joining.