Telomerase-associated Protein 1, HSP90, and Topoisomerase IIα Associate Directly with the BLM Helicase in Immortalized Cells Using ALT and Modulate Its Helicase Activity Using Telomeric DNA Substrates

Abstract

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 ...

FE65 regulates and interacts with the Bloom syndrome protein in dynamic nuclear spheres – potential relevance to Alzheimer's disease

“…Finally tetraploidy, a putative initial result of the re-entry is also a known AD feature (Frade and López-Sánchez, 2010). Notably, BLM colocalizes with TERF2 (telomeric repeat-binding factor 2) in foci actively synthesizing DNA during late S and G2/M phases of the cell cycle, which has been described for immortalized cells using the alternative lengthening of telomere pathway (Bhattacharyya et al, 2009;Yankiwski et al, 2000). TERF2 was also identified as a differentially abundant protein in FE65 knockdown cells supporting a role for FE65 in cell cycle regulation.…”

“…BLM is a DNA helicase present in nuclear foci that are sites of DNA synthesis during the late S and G2/M phases of the cell cycle (Bhattacharyya et al, 2009). The MCM (minichromosome maintenance) protein family plays an important role in S-phase genome stability in the context of DNA replication, damage and repair (Bailis and Forsburg, 2004;Mincheva et al, 1994).…”

Inhibition of NF-κB by combination therapy with parthenolide and hyperthermia and kinetics of apoptosis induction and cell cycle arrest in human lung adenocarcinoma cells

“…The reason why agents such as AMR, AMROH, and BLM cause no cell phase responses may be due to the fact that they target different types of DNA damage (40)(41)(42). As for the mechanisms of those agents, it has been reported that BLM predominantly co-localizes with 3 proteins that bind directly to BLM, telomerase-associated protein 1, heat shock protein 90, and topoisomerase II ·, in foci actively synthesizing DNA during the late S and G 2 /M phases of the cell cycle in L cells (43), while AMR and AMROH inhibit cell growth prior to DNA-protein complex formation followed by double-strand DNA breaks, which are mediated by topoisomerase II (44). Heat induced apoptosis was also significantly increased by combinations of those agents.…”

Two genetic variants in telomerase-associated protein 1 are associated with stomach cancer risk

“…28 However, TEP1, heat-shock protein 90 and topoisomerase IIα associate directly with bloom syndrome protein (BLM) helicase in immortalized cells using the alternative lengthening of telomere (ALT) pathway and modulate its helicase activity. 29 Cells deficient in BLM helicase display increased telomere association between homologous chromosomes. BLM helicase is important for proper telomere maintenance and function in ALT cells.…”