Telomerase-associated protein 1, HSP90, and topoisomerase IIalpha associate directly with the BLM helicase in immortalized cells using ALT and modulate its helicase activity using telomeric DNA substrates.

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 IIalpha (TOPOIIalpha). 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 TOPOIIalpha are components of a specific complex containing BLM, TRF1, and TRF2 but that this complex does not include TEP1. TEP1, TOPOIIalpha, and HSP90 interact directly with BLM in vitro and modulate its helicase activity on telomere-like DNA substrates but not on non-telomeric substrates. Initial studies suggest that knockdown of BLM in ALT cells reduces average telomere length but does not do so in cells using telomerase.

Deriving mouse spermatogonial stem cell lines.

In adult males, spermatogonial stem cells function to replenish developing gametes that are continuously released from the testes as mature spermatozoa. Because of their potential importance to research, medicine, industry, and conservation, numerous attempts have been made in the past to cultivate sperma-togonial stem cells in vitro. However, only recently have culture methods been established that effectively promote the proliferation of mammalian spermatogonial stem cells in vitro. We describe a simple and reproducible protocol for the derivation and maintenance of mouse spermatogonial stem cell lines that proliferate for long periods of time in culture.

BLM helicase complements disrupted type II telomere lengthening in telomerase-negative sgs1 yeast.

Recombination-mediated pathways for telomere lengthening may be utilized in the absence of telomerase activity. The RecQ-like helicases, BLM and Sgs1, are implicated in recombination-mediated telomere lengthening in human cells and budding yeast, respectively. Here, we show that BLM expression rescues disrupted telomere lengthening in telomerase-negative sgs1 yeast. BLM helicase activity is required for this complementation, indicating BLM and Sgs1 resolve the same telomeric structures. These data support a conserved function for BLM and Sgs1 in recombination-mediated telomere lengthening.

Association and regulation of the BLM helicase by the telomere proteins TRF1 and TRF2.

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.