The roles of WRN and BLM RecQ helicases in the Alternative Lengthening of Telomeres

Abstract

Approximately 10% of all cancers, but a higher proportion of sarcomas, use the recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. Two RecQ helicase genes, BLM and WRN, play important roles in homologous recombination repair and they have been implicated in telomeric recombination activity, but their precise roles in ALT are unclear. Using analysis of sequence variation present in human telomeres, we found that a WRN- ALT+ cell line lacks the class of complex telomere mutations attributed to inter-telomeric recombination in other ALT+ cell lines. This suggests that WRN facilitates inter-telomeric recombination when there are sequence differences between the donor and recipient molecules or that sister-telomere interactions are suppressed in the presence of WRN and this promotes inter-telomeric recombination. Depleting BLM in the WRN- ALT+ cell line increased the mutation frequency at telomeres and at the MS32 minisatellite, which is a marker of ALT. The absence of complex telomere mutations persisted in BLM-depleted clones, and there was a clear increase in sequence homogenization across the telomere and MS32 repeat arrays. These data indicate that BLM suppresses unequal sister chromatid interactions that result in excessive homogenization at MS32 and at telomeres in ALT+ cells.

Figure 1.

Characterization of Xp/Yp telomere mutations in the W-V cell line and clones transfected with the shBLM or control plasmid. To identify mutations that occur at a low frequency in a population of cells, single telomere molecules that had been amplified by STELA were analysed by TVR-PCR from (A) the W-V cell line, (B) in two clones expressing the shBLM plasmid and (C) in a W-V control clone transfected with the empty vector. The interspersion of TTAGGG repeats (T) and the sequence-variant TGAGGG repeats (G) are shown for the progenitor allele and the mutant molecules. The region(s) where the mutant molecule differs from the progenitor allele is underlined. The number of times each telomere map was observed among the molecules analysed is shown in brackets. The TVR maps for some of the W-V-shBLM clone 8 mutant molecules (in italics) are shown in Supplementary Figure 4. As an example, the block of nine T-type repeats in bold in W-V-shBLM clone 6 mutant 1 identifies the region where the mutation was initiated.

Figure 2.

Reduction of BLM expression reduces colony formation in ALT+ cell lines. (A) Colony forming assays in the U2OS and W-V ALT+ cell lines. Cells were transfected with the shBLM or empty vector (ctrl) plasmids and after two weeks in selective medium the plates were fixed, stained and colonies counted. The histograms show the results from three transfection experiments, in which 5 × 10⁵ cells were transfected with 3 µg of plasmid. The error bars show standard error of the mean (SEM). (B) western blot analysis of clones isolated for further analysis. The W-V parental cell line, two control clones (empty vector) and nine clones transfected with the shBLM-plasmid are shown. Among the shBLM clones, only clones 6 and 8 show reduced BLM expression.

Figure 3.

Small-pool PCR detection of MS32 length mutant molecules in shBLM-plasmid and control clones. The tracks show the results of MS32 amplification from small aliquots of DNA followed by detection by Southern blot hybridization. The common or more stable alleles in each clone are present in all tracks across the gel and identified with arrows in W-VshBLM clone 6. Mutant molecules that differ in size from the progenitor bands were counted.

Figure 4.

Partial characterization of MS32 mutant molecules in the W-V parental cell line and W-VshBLM clone 6. The order of MS32 sequence-variant repeats was determined by MVR-PCR from the flanking sequence at each end of the repeat array. The complete MVR maps are shown for the three common alleles in the W-V cell line, and comparison suggests that allele 2 was derived from allele 1. MVR maps are shown for seven different length mutant molecules from the W-V cell line and five from the W-V-shBLM clone 6. It was not possible to determine the complete MVR maps for long molecules and so gaps are shown ( … ) to indicate missing data. The four sequence-variant repeats within the MS32 minisatellite are Y, y, e and E (49). The location of the germline recombination hotspot, adjacent to one end of the minisatellite, is indicated with an arrow.

Figure 5.

Model for the roles of WRN and BLM at telomeres and MS32 in ALT+ cells. (A) In cells that utilize the ALT pathway, telomeres can be extended by strand invasion and copying from a non-homologous telomere by a BIR mechanism that is promoted by WRN (lower arrow). In ALT+ cells, double strand breaks at MS32 (and sometimes telomeres) can be repaired via SDSA, which may involve multiple rounds of strand invasion between misaligned sister chromatids, copying and disassociation (upper arrow). However, BLM and perhaps WRN suppress outcomes that result in repeat sequence homogenization (through homology dependent deletions (HDD) or BIR-type events). (B) In the WRN negative ALT+ (W-V) cell line, BIR between non-homologous (non identical) telomeres is reduced or absent (lower arrow). Suppression of double-strand break repair that leads to HDD or BIR-type events between misaligned sister chromatids at MS32 and at telomeres is relaxed, possibly because WRN is absent or suppression by BLM is relaxed, and mutations that result in homogenization of the repeat arrays are seen. (c) Down-regulation of BLM in a WRN negative ALT+ cell line further reduces suppression of the events between sister chromatids at MS32 and at telomeres and the frequency of mutant molecules that show repeat homogenization increases.