In addition to increased DNA-strand exchange, a cytogenetic feature of cells lacking the RecQ-like BLM helicase is a tendency for telomeres to associate. We also report additional cellular and biochemical evidence for the role of BLM in telomere maintenance. BLM co-localizes and complexes with the telomere repeat protein TRF2 in cells that employ the recombination-mediated mechanism of telomere lengthening known as ALT (alternative lengthening of telomeres). BLM co-localizes with TRF2 in foci actively synthesizing DNA during late S and G2/M; co-localization increases in late S and G2/M when ALT is thought to occur. Additionally, TRF1 and TRF2 interact directly with BLM and regulate BLM unwinding activity in vitro. Whereas TRF2 stimulates BLM unwinding of telomeric and non-telomeric substrates, TRF1 inhibits BLM unwinding of telomeric substrates only. Finally, TRF2 stimulates BLM unwinding with equimolar concentrations of TRF1, but not when TRF1 is added in molar excess. These data suggest a function for BLM in recombination-mediated telomere lengthening and support a model for the coordinated regulation of BLM activity at telomeres by TRF1 and TRF2.

Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre-and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers. Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations. BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family. To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase. The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction. Cell extracts deficient in BLM were competent for DNA mismatch repair. These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair.Bloom's syndrome (BS) 1 is a rare autosomal recessive disorder characterized by immunodeficiency, short stature, male infertility, and an increased risk of a broad spectrum of cancers (1). Cells isolated from BS individuals are characterized by cytogenetic abnormalities, with the hallmark feature of hyperrecombination between sister chromatids. BS chromosomes also display increased levels of breaks, translocations, quadriradial formations, and telomeric associations (2).The gene mutated in BS was positionally cloned and named BLM; it encodes a 1417-amino acid protein with strong homology to the Escherichia coli RecQ family of DNA and RNA helicases (3). The E. coli RecQ helicase participates in homologous recombination and suppresses illegitimate recombination (4, 5). Other eukaryotic RecQ family members include Sgs1p from Saccharomyces cerevisiae and Rqh1p from Schizosaccharomyces pombe; loss of function of either of these helicases results in genomic instability (6, 7). Mutations in other human RecQ helicases result in the rare autosomal recessive disorders Werner's syndrome and Rothmund-Thomson syndrome, also characterized by chromosomal instability and cancer predisposition (8, 9).The BLM helicase unwinds duplex DNA from 3Ј to 5Ј in the presence of ATP (10, 11). It also selectively recognizes and promotes branch migration of Holliday junctions in vitro (12). BLM can be found in a large protein complex in the nucleus with other proteins involved in DNA repair such as BRCA1, ATM, MLH1, MSH2, MSH6, and replication factor C (13). However, direct interactions of BLM have only been demonstrated biochemically with replication protein A (RPA) and topoisomerase III␣ (14 -16). These experiments suggest that the BLM helicase interacts with a variety of nuclear proteins to perform functions in DNA replication, recombination, or repair.To understand the role of the BLM helicase in maintaining genomic stability, a yeast two-hybrid screen was used to identify...

The BLM helicase associates with the telomere structural proteins TRF1 and TRF2 in immortalized cells using the alternative lengthening of telomere (ALT) pathways. This work focuses on identifying protein partners of BLM in cells using ALT. Mass spectrometry and immunoprecipitation techniques have identified three proteins that bind directly to BLM and TRF2 in ALT cells: telomerase-associated protein 1 (TEP1), heat shock protein 90 (HSP90), and topoisomerase II␣ (TOPOII␣). BLM predominantly co-localizes with these proteins in foci actively synthesizing DNA during late S and G 2 /M phases of the cell cycle when ALT is thought to occur. Immunoprecipitation studies also indicate that only HSP90 and TOPOII␣ are components of a specific complex containing BLM, TRF1, and TRF2 but that this complex does not include TEP1. TEP1, TOPOII␣, and HSP90 interact directly with BLM in vitro and modulate its helicase activity on telomere-like DNA substrates but not on nontelomeric substrates. Initial studies suggest that knockdown of BLM in ALT cells reduces average telomere length but does not do so in cells using telomerase. Bloom syndrome (BS)4 is a genetic disease caused by mutation of both copies of the human BLM gene. It is characterized by sun sensitivity, small stature, immunodeficiency, male infertility, and an increased susceptibility to cancer of all sites and types. The high incidence of spontaneous chromosome breakage and other unique chromosomal anomalies in cells from BS patients indicate an increase in homologous recombination in somatic cells (1). Another notable feature of non-immortalized and immortalized cells from BS individuals is the presence of telomeric associations (TAs) between homologous chromosomes (2). Work from our group and others have suggested a role for BLM in recombination-mediated mechanisms of telomere elongation or ALT (alternative lengthening of telomeres), processes that maintain/elongate telomeres in the absence of telomerase (3-5). However, the exact mechanism by which BLM contributes to telomere stability is unknown.Several proteins interact with and regulate BLM helicase activity, including two telomere-specific proteins, TRF1 and TRF2 (6, 7). Although TRF2 stimulates BLM unwinding of telomeric and non-telomeric 3Ј-overhang substrates, TRF1 inhibits BLM unwinding of telomeric substrates. TRF2-mediated stimulation of BLM helicase activity on a telomeric substrate is observed when TRF2 is present in excess or with equimolar amount of TRF1 but not when TRF1 is present in molar excess. Both proteins associate with BLM specifically in ALT cells in vivo, suggesting their involvement in the ALT pathways. In addition to TRF1 and TRF2, the telomere single-strand DNAbinding protein POT1 strongly stimulates BLM helicase activity on long telomeric forked duplexes and D-loop structures (8).Other proteins also play an important role in telomere maintenance in telomerase-negative cells, including RAD50, NBS1, and MRE11, which co-localize with TRF1 and TRF2 in specialized ALT-associated promyelocytic ...