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 IIα
(TOPOIIα). 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 TOPOIIα are components of a specific complex
containing BLM, TRF1, and TRF2 but that this complex does not include TEP1.
TEP1, TOPOIIα, 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.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 3′-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 DNA-binding 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 leukemia (PML)
nuclear bodies (APBs)
(
9–
11).
Thus, we hypothesize that BLM complex formation may be essential for the ALT
mechanism, and its modification may occur dynamically during the specific
nucleic acid transactions required to protect the telomere in cells using the
ALT pathways.This study has identified previously unknown protein partners of BLM and
TRF2 in ALT cells using double immunoprecipitation and mass spectrometry (MS).
These include telomerase-associated protein 1 (TEP1), heat shock protein 90
(HSP90), and topoisomerase IIα (TOPOIIα). These proteins associate
with BLM and TRF2 in cells using ALT but not in cells using telomerase and
directly interact with BLM
in vitro. This complex of proteins
localizes to sites of new DNA synthesis
in vivo in ALT cells,
suggesting a role in telomere maintenance. We also identified HSP90 and
TOPOIIα in another ALT-specific complex consisting of BLM, TRF1, and
TRF2 but not TEP1.
In vitro analyses demonstrate that HSP90 inhibits
BLM helicase activity using both telomeric and non-telomeric substrates,
whereas TEP1 and TOPOIIα initially slow the kinetics of BLM unwinding
only using telomeric substrates. These findings suggest the presence of
dynamic BLM-associated ALT complexes that include previously unidentified
interacting proteins. The function of TEP1 in the BLM·TRF2 complex
remains unclear, although its previously described interaction with the RNA
subunit of telomerase (
12)
suggests an interesting hypothesis of cross-talk between mechanisms of
telomere elongation.
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