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

Abstract

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.

Figure 1.

Telomere dynamics and fusion in ATLD4 cells. (A) XpYp and 17p STELA, each sample is analysed with four separate reactions; the population doubling (PD) point of the culture is indicated above each gel. Mean telomere length, standard deviation and rate of telomere erosion as a function of PD is indicated below the gel. (B) Telomere fusion analysis, single molecule fusion events are detected by southern hybridization with the XpYp and 17p telomere-adjacent probes as indicated on the right. The PD points of the cultures are indicated above.

Figure 2.

Illustrating the mutational profile that accompanies telomere fusion in ATLD 3/4 cells. (A) The frequency of TTAGGG repeats at the fusion point. (B) Micro-homology at the fusion point. (C–E) Representing the distance of sub-telomeric deletion from the start of the telomere repeat arrays of 17p, XpYp and 21q. The extent of sub-telomeric deletion that can be detected with the assays is indicated by a dotted line across the x-axis. The means ± standard error are indicated on each panel. (F) Pie chart displaying the proportions of telomere fusion events displaying sequence homology, nucleotide insertions and identifiable insertions at the fusion point in MRC5 and ATLD 3/4 cells.

Figure 3.

Displaying the DNA sequence at the fusion point in a subset of fusions that display nucleotide insertions in ATLD cells (A) and MRC5 cells (B). The fusion sequence is displayed on the top lines, with the fusion partners displayed below. Arrows indicate the fusion point, the DNA between the arrows represent the DNA insertions, the participating chromosomes and the deletion sizes are stated above, asterisks below indicate homology between the fusion partners, sequence highlighted in red and underlined indicate duplicated sequences across the fusion point. No duplications could be detected in fusions 13 and 15.

Figure 3.

Displaying the DNA sequence at the fusion point in a subset of fusions that display nucleotide insertions in ATLD cells (A) and MRC5 cells (B). The fusion sequence is displayed on the top lines, with the fusion partners displayed below. Arrows indicate the fusion point, the DNA between the arrows represent the DNA insertions, the participating chromosomes and the deletion sizes are stated above, asterisks below indicate homology between the fusion partners, sequence highlighted in red and underlined indicate duplicated sequences across the fusion point. No duplications could be detected in fusions 13 and 15.