BLM Helicase Complements Disrupted Type II Telomere Lengthening in Telomerase-Negative sgs1 Yeast

Bloom's syndrome is a rare autosomal recessive disorder characterized by a high predisposition to cancer of many tissues and types. Somatic cells display extraordinary chromosome instability with a high frequency of sister chromatid exchanges, chromosome configurations signifying recombination, and telomeric associations. BLM, the gene mutated in Bloom's syndrome, encodes a RecQ-like helicase that unwinds displacement loops, G-quadruplexes, and promotes the branch migration and resolution of Holliday junctions in vitro (1–3). The Saccharomyces cerevisiae genome encodes only one RecQ family member, SGS1, the deletion of which results in excessive sister chromatid exchanges and interchromosomal recombination reminiscent of the Bloom's syndrome phenotype (4). BLM can also suppress hyperrecombination and premature aging of sgs1 mutants (5). Similar to BLM, Sgs1 unwinds G-quadruplexes and synthetic Holliday junctions in vitro (6, 7). Furthermore, BLM and Sgs1 share a conserved interaction with topoisomerase IIIα, which may facilitate non-crossover resolution of Holliday junctions (8, 9).

In the absence of telomerase, immortalized mammalian cells and yeast may utilize recombination-mediated pathways to maintain telomeres, termed alternative lengthening of telomeres (ALT), in mammalian cells. In human cells using ALT, BLM associates with telomeres and the telomere repeat factors TRF1 and TRF2 (10, 11). Overexpression of BLM in cells using ALT increases telomeric signals, suggesting that BLM promotes ALT (10). In addition to BLM, the related RecQ-like Werner's syndrome helicase, WRN, as well as Rad52, Rad51, RPA, and the Rad50/Mre11/Nbs1 complex, associate with telomeric foci in cells using ALT (12, 13). Telomerase-negative S. cerevisiae overcome telomere crisis by utilizing one of two Rad52-dependent recombination-mediated pathways, termed type I and type II (14). Type I telomere lengthening requires Rad51, Rad54, and Rad57, whereas type II telomere lengthening requires Rad50, Rad59, and Sgs1 (12, 15–17). C_1-3A/TG_1-3 terminal repeats in type II survivors are amplified and often heterogeneous in length, whereas type I survivors have amplified subtelomeric Y′-elements and very short tracts of C_1-3A/TG_1-3 terminal repeats (Fig. 1A). Like type II survivors, terminal telomeric repeats in human cells using ALT are long and heterogeneous, suggesting that these pathways involve similar mechanisms (18). The association of BLM and Sgs1 with the ALT and type II pathway, respectively, led us to hypothesize a conserved function of these RecQ helicases in recombination-mediated telomere lengthening.

Expression vectors. pYES-BLM expression construct (pJK1; ref. 19) was provided by I. Hickson (University of Oxford, Oxford, United Kingdom). The conserved lysine at amino acid 695 of the human BLM protein was mutated to glutamic acid by PCR-mediated site-directed mutagenesis of the BLM gene within the pYES-BLM vector to create pYES-K695E BLM. The presence of the mutation was confirmed by sequencing. Helicase assays with purified proteins expressed from pYES vector and purified from yeast confirmed unwinding activity of wild-type BLM (11) and lack of unwinding with K695E-BLM (data not shown).

Yeast strains and crosses. sgs1::HIS3; URA3 yeast (RDKY3813; ref. 20) was provided by R. Kolodner (University of California-San Diego, La Jolla, CA). The URA3 marker present in this strain was eliminated by selection on 5-FOA, confirmed by PCR and lack of growth on media lacking uracil, to create sgs1::HIS3, which was used for all subsequent experiments. est2::KANMX4/EST2 (BY4743-YLR318W) was obtained from American Type Culture Collection Yeast Genetic Stock Center, Manassas, VA. The latter was streaked onto standard sporulation media to obtain MATα; est2::KANMX4 spores, which were mated to MATa; sgs1::HIS3 to produce a double-heterozygous diploid strain that was subsequently transformed with pYES-BLM, pYES-K695E, or pYES vector (Invitrogen, Carlsbad, CA). The diploid strain was sporulated and tetrads dissected. Individual spores with genotypes SGS1 EST2, sgs1::HIS3 EST2, SGS1 est2::KANMX4, and sgs1::HIS3 est2::KANMX4 were selected on the appropriate media and passaged on uracil dropout plates with minimal base containing 2% galactose at 30°C. After restreaking 10 times (∼250 cell generations), single colonies were selected and grown in standard liquid uracil dropout medium with minimal base containing 2% galactose. Yeast were diluted 1:1,000 every 24 to 72 hours (when cultures reached ∼1 × 10⁸ cells/mL) into fresh media for an additional 10 passages (∼100 cell generations) and were subsequently collected for telomere analysis.

Telomere Southern blot analysis. Yeast were pelleted by centrifugation, resuspended in 200 μL lysis buffer [1% (w/v) SDS, 50 mmol/L EDTA, 0.1 mol/L Tris (pH 8.0)] with acid-washed glass beads, and lysed by vortexing for 2 minutes. Genomic DNA was extracted with phenol/chloroform, ethanol-precipitated, and resuspended in Tris-EDTA buffer. Genomic DNA (7.5 μg) was digested with XhoI (New England Biolabs, Beverly, MA) at 37°C overnight and resolved by electrophoresis on a 1% Tris-borate EDTA agarose gel. Digested DNAs were transferred onto a positively charged Hybond-N+ nylon membrane (Amersham Biosciences, Piscataway, NJ) and probed by standard Southern blotting procedures. A probe for the TG_1-3 repeats was created by random priming with [α-³²P]dCTP using poly(dA-dC)·poly(dG-dT) template (Amersham Biosciences) as described by others (12). Southern blots were developed and analyzed by autoradiography.

The Sgs1 helicase is required for the type II pathway such that telomerase-negative sgs1 mutants give rise to type I survivors exclusively (12, 16, 17). To test whether BLM and Sgs1 have conserved functions in recombination-mediated telomere lengthening, rescue of the type II pathway by BLM expression was tested in yeast lacking expression of Sgs1 and Est2, the catalytic subunit of telomerase. sgs1::HIS3/SGS1; est2::KAN2/EST2 diploid yeast were transformed with empty expression vector or expression vector encoding BLM or K695E-BLM. K695E-BLM, created by site-directed mutagenesis, encodes a BLM protein that is mutated within the conserved ATP-binding site resulting in loss of helicase activity (21). The diploid strain was sporulated and tetrads were dissected to identify individual sgs1 est2, SGS1 est2, sgs1 EST2, and SGS1 EST2 (wild-type) spores from which individual survivor colonies were established and subsequently cultured in liquid media. DNAs were isolated and digested with XhoI to release the C_1-3A/TG_1-3 terminal repeats, the terminal restriction fragment (TRF), from the subtelomeric 6.7 and 5.2 kb Y′-elements (Fig. 1B). Digested DNAs were analyzed by Southern blot hybridization with a probe that identifies TG_1-3 repeats. The separation of Y′-elements by short tracts of C_1-3A/TG_1-3 allows identification of Y′-elements and TRFs simultaneously.

Southern blot analysis of telomeres is shown in Fig. 1C. Seven of eight SGS1 est2 survivors transformed with vector only (est2 + vector) utilized the type II pathway, identified by the presence of TRFs of heterogeneous distribution (type II-TRFs). A single type I SGS1 est2 survivor was identified by the presence of intense Y′-element bands and a short TRF fragment (Y′-TRF). SGS1 est2 survivors expressing BLM utilized type I and type II pathways as well, although a smaller number of type II survivors were identified compared with SGS1 est2 yeast transformed with empty vector (data not shown). sgs1 est2 yeast transformed with empty vector (sgs1 est2 + vector) gave rise to type I survivors only, consistent with the requirement of Sgs1 for type II telomere lengthening. In contrast, BLM expression induced in sgs1 est2 yeast (sgs1 est2 + BLM) resulted in the appearance of three type II survivors out of eight cultures, demonstrating that BLM can rescue the type II telomere lengthening defect associated with the absence of Sgs1 in some cultures. Finally, all sgs1 est2 survivors expressing a helicase-dead BLM mutant (sgs1 est2 + K695E BLM) utilized the type I pathway, demonstrating that BLM helicase activity is required for rescue of the type II pathway.

Our experiments show that BLM can complement disrupted type II telomere lengthening in sgs1 est2 mutants. Complementation requires BLM helicase activity, indicating that BLM and Sgs1 resolve common homologous structure(s) to promote type II telomere lengthening. Given that BLM promotes telomere signal amplification in human cells using ALT (10), these data are consistent with a conserved function of BLM and Sgs1 in recombination-mediated telomere lengthening in mammalian and yeast cells, respectively. Furthermore, these data support the hypothesis that ALT and the type II pathway extend telomeres by a common mechanism.

Three of eight sgs1 est2 survivors expressing BLM utilized the type II pathway compared with seven of eight SGS1 est2 survivors transformed with vector alone. Rescue of the type II pathway by BLM may be partial due to differences in pYES-BLM copy number, and thus variable BLM expression in individual cultures. Notably, when BLM expression was induced in SGS1 est2 yeast, only two of eight survivors utilized the type II pathway (data not shown). Therefore, it is likely that very high levels of BLM expression in some cultures can interfere with normal functioning of the type II machinery by a dominant-negative effect of overexpression that results in a lower than expected number of type II survivors.

The precise mechanisms by which recombination-mediated telomere lengthening occur and the functions of BLM and Sgs1 in these pathways remain speculative. The preferential unwinding of branched DNA substrates by these helicases in vitro (6, 22) suggests the possibility that they process recombination intermediate(s), such as Holliday junctions and displacement loops, at telomeres undergoing recombination. Additionally, these RecQ-like helicases may be required to unwind secondary structures, such as G-quadruplex DNA, that occur during recombination within the GC-rich C_1-3A/TG_1-3 terminal repeats in yeast and the TTAGGG_n telomeric repeats in mammalian cells. G-quadruplex DNA is readily unwound by Sgs1, BLM, and WRN helicases in vitro (1, 7, 22). WRN can also partially rescue the type II pathway in sgs1 est2 mutants and colocalizes with telomeric proteins in human cells using ALT (12, 16), although its contribution to ALT remains unknown. An inability to unwind G-quadruplexes may prohibit replication of the lengthy C_1-3A/TG_1-3 terminal repeats in the absence of Sgs1 such that the type I pathway is required for prolonged survival without telomerase. Notably, no comparable type I mechanism has been uncovered in telomerase-negative mammalian tumors or cell lines. By extension, cells lacking BLM, or possibly WRN, may be unable to utilize ALT in the absence of telomerase, predicting that all tumors and stem cell populations in persons with Bloom's or Werner's syndrome use telomerase for telomere maintenance.

Grant support: National Institute for Environmental Health Sciences grant P30-ES06096.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. I. Hickson and R. Kolodner for supplying reagents and Dr. Y. Sanchez for experimental assistance.