Cooperation of DNA-PKcs and WRN helicase in the maintenance of telomeric D-loops
Abstract
Werner syndrome is an inherited human progeriod syndrome caused by mutations in the gene encoding the Werner Syndrome protein, WRN. It has both 3'-5' DNA helicase and exonuclease activities, and is suggested to have roles in many aspects of DNA metabolism, including DNA repair and telomere maintenance. The DNA-PK complex also functions in both DNA double strand break repair and telomere maintenance. Interaction between WRN and the DNA-PK complex has been reported in DNA double strand break repair, but their possible cooperation at telomeres has not been reported. This study analyzes thein vitro and in vivo interaction at the telomere between WRN and DNA-PKcs, the catalytic subunit of DNA-PK. The results show that DNA-PKcs selectively stimulates WRN helicase but not WRN exonuclease in vitro, affecting that WRN helicase unwinds and promotes the release of the full-length invading strand of a telomere D-loop model substrate. In addition, the length of telomeric G-tails decreases in DNA-PKcs knockdown cells, and this phenotype is reversed by overexpression of WRN helicase. These results suggest that WRN and DNA-PKcs may cooperatively prevent G-tail shortening in vivo.
Introduction
Werner syndrome (WS) is a hereditary disorder
associated with symptoms of premature aging, including early onset of
cataracts, osteoporosis, atherosclerosis and cancer [1,2]. The cellular phenotype of WS
includes premature cellular senescence, telomere dysfunction and chromosome
instability. WS is caused by mutations in the gene encoding the Werner
syndrome protein (WRN), a multifunction protein that possesses 3'-5' DNA
helicase, 3'-5' DNA exonuclease, branch migration, and strand annealing
activities [3-8]. WRN helicase is active on a
wide variety of DNA substrates, with preference for forked duplex molecules and
structures at telomeric DNA [9].
Telomeres
are nucleoprotein structures at the ends ofeukaryotic chromosomes.
In humans, telomeric DNA includes a duplex region containing tandem repeats of
the sequence 5'-TTAGGG-3' and telomeric 3'-G-overhang, so called G-tail. The
telomere DNA loops back on itself forming a lariat t-loop structure, where the
G-tail invades the duplex telomeric repeats and forms a D loop (displacement
loop) that stabilizes the t-loop [10]. A complex
of six human telomere binding proteins, called shelterin, has been identified [11]. These
include TRF1, TRF2, TIN2, RAP1, TPP1 and POT1. Shelterin promotes formation of
a t-loop, which is critical for protecting the G-tail and maintaining telomere
length and structure. WRN has also been detected in telomere complexes. It
interacts with TRF2 and POT1, and regulates telomere processing during S phase [12-14]. This
WRN function is biologically important, because WS fibroblasts display
accelerated telomere erosion and stochastic telomere loss [15], and WS
lymphoblasts show erratic telomere length dynamics [15-17]. The
DNA-PK complex, which is composed of a catalytic subunit, DNA-PKcs, regulatory
subunits Ku70, and Ku80, is a DNA damage sensing serine-threonine protein
kinase that is critical for repair of DNA double strand breaks. This complex
was found at telomeres and DNA-PKcs-deficient cells also exhibit dysfunctional
telomeres [18,19]. In
addition to the similar defects of telomere in WS cells and DNA-PKcs deficient
cells, DNA-PKcs interacts with and phosphorylates WRN in response to DNA
double-strand breaks [20-22]. Thus,
these two proteins may also cooperate in telomere metabolism.
Figure 1. D-loop unwinding by WRN in the absence and presence of DNA-PKcs. (A) The
D-loop substrate consisted with INV, BT and BB. 5'-end of INV was
radiolabeled as indicated by asterisk. WRN (3.3 nM, lanes 3-6) and
increasing amounts of DNA-PKcs (0.67 nM, lane 4; 3.3 nM, lanes 5 and 7;
16.7 nM, lane 6) were incubated in standard reaction buffer prior to
addition of the telomeric D-loop substrate. Reaction products were
analyzed by native (B) or denaturing gel electrophoresis (C).
Lanes 1 in (B) and (C): A DNA ladder marker.
Here,
we report findings that add novel insight into the function of WRN and DNA-PKcs
at telomeres. DNA-PKcs stimulates WRN helicase activity on D-loop substrates.
Measurements of telomere length revealed that G-tail shortenings in DNA-PKcs-deficient cells were reversed by overexpression
of WRN helicase. We propose that the DNA-PKcs and WRN cooperation play a
critical and interactive role in maintaining telomere length and structure in
proliferating cells.
Results
DNA-PKcs
modulates WRN processing of telomeric D-loops
The
effect of DNA-PKcs on WRN was analyzed using an in vitro telomeric
D-loop unwinding assay. The DNA substrate used in this assay consists of a
bubble with two 30 bp duplex arms separated by a 33 nt ssDNA "melted"
region, one strand of which is annealed to an "invading" ssDNA (INV)
(Figure 1A). The melted region and the invading ssDNA carry telomeric repeats,
such that the DNA substrate mimics a telomeric D-loop. Previous studies with
this DNA substrate showed that WRN exonuclease partially degrades and the WRN
helicase unwinds and releases the INV DNA strand, which is stable after release
because WRN exonuclease does not efficiently degrade ssDNA [12]. In this study, the DNA substrate was incubated with WRN in
the absence or the presence of increasing
amounts of DNA-PKcs. Under these conditions, WRN was not phosphorylated by the
DNA-PKcs since Ku70 and Ku80 are absent. Reaction products were analyzed by
native and denaturing gel electrophoresis, as shown in Figures 1B and 1C,
respectively. In the absence of PKcs, WRN released 52- and 46-mer ssDNA
products (Figure 1B and 1C, lanes 3), consistent with its pausing at the GGG
sequence in the telomeric repeat, as reported previously [12]. In the
presence of up to a 5-fold molar excess of DNA-PKcs to WRN, the ssDNA reaction
products were longer, primarily 52-, 58-, and 64- nucleotides in length
(Figures 1B and 1C, lanes 6). However, the total ssDNA product (and the amount
of unreacted DNA substrate) was similar in WRN reactions with or without
DNA-PKcs (Figures 1B and 1C, lanes 6). These results suggested two possibilities;
i) the processivity of WRN exonuclease is inhibited by DNA-PKcs, or ii) the
processivity of WRN helicase is stimulated by DNA-PKcs.
DNA-PKcs
stimulates WRN helicase activity on telomeric D-loops
The
effect of DNA-PKcs on WRN enzymatic functions was examined by incubating WRN
with telomeric D-loop substrates in the absence of ATP, which inactivates WRN
helicase without affecting WRN exonuclease. Under these conditions, WRN
exonuclease produced 64-, 58-, 52- and 46-mer reaction products, and the
distribution of reaction products was unchanged by addition of DNA-PKcs (Figure 2A). Thus, DNA-PKcs does not inhibit WRN exonuclease. The ability of DNA-PKcs
to stimulate WRN helicase activity was examined by incubating an
exonuclease-deficient point mutant, WRN (E84A) with telomeric D-loop substrates
in the absence or presence of DNA-PKcs. WRN (E84A), which has a normal level
of helicase activity but no exonuclease activity, unwinds 3.3% of the telomeric
D-loop substrate in the absence of DNA-PKcs (Figure 2B, lane 3) and unwinds 66%
of the substrate in the presence of
DNA-PKcs, producing a full-length INV (Figure 2B, lane 4). This very significant
stimulation is not observed in reactions containing Ku 70/80 (Figure 2B, lane
5), arguing against the possibility that a low level contamination of DNA-PKcs
with Ku is responsible for the observed stimulation of WRN helicase. These
results suggest that DNA-PKcs stimulates WRN helicase, possibly by increasing
its processivity, and that this stimulation is independent of WRN exonuclease.
DNA-PKcs
does not stimulate BLM helicase activity on telomeric D-loops
BLM
is a human RecQ family helicase, which like WRN, is proposed to play a role at
telomeres in human cells [14].
Therefore, the effect of DNA-PKcs on BLM ability to unwind telomeric D-loop DNA
substrates was examined (Figure 3). In reactions containing a low
concentration of BLM, BLM failed to unwind the telomeric D-loop in the absence
or presence of DNA-PKcs. However, when replication protein A (RPA) was added
to the same amount of BLM, BLM helicase fully unwound the telomeric D-loop,
producing full-length INV, as previously reported [13]. Thus,
DNA-PKcs does not stimulate BLM helicase, indicating that its interaction with
WRN helicase is specific.
Figure 2. Differential Effect of DNA-PKcs on WRN helicase and
exonuclease activities. (A) WRN (3.3 nM, lanes 3-5) and DNA-PKcs
(3.3 nM, lane 4; 16.7 nM, lanes 5 and 6) were incubated in standard reaction
buffer lacking ATP prior to addition of the D-loop substrate. Reaction products
were analyzed by denaturing gel electro-phoresis. Lanes 1 and 7: A DNA ladder
marker. (B) WRN (E84A) (3.3 nM, lanes 3-5) was preincubated with either
DNA-PKcs (16.7 nM, lane 4) or Ku (3.3 nM, lane 5) in standard reaction buffer
prior to addition of the D-loop substrate. Reaction products were analyzed by
native gel electrophoresis. Lane 1: heat-denatured D-loop substrate denoted by
a filled triangle. Lane 6: A DNA ladder marker.
DNA-PKcs
stimulates WRN helicase activity on non- telomeric D-loops
The ability of DNA-PKcs to stimulate WRN
wild type or WRN (E84A) helicase on non-telomeric D-loop substrates was also
examined (Figure 4). The results show that wild type and WRN (E84A) unwinds a
small fraction of the non-telomeric D-loop substrate in the absence of
DNA-PKcs, and the addition of DNA-PKcs increased the unwinding, while it
enabled WRN to produce longer ssDNA products (Figure 4, lanes 9-13). Similar
results were observed with telomeric D-loop DNA substrates, as observed in Figures
1B and 2B (Figure 4, lanes 2-6). These results suggest that DNA-PKcs may
stimulate WRN helicase activity on D-loop structures in telomeric or
non-telomeric DNA because the stimulation appears to be independent of the
nucleotide sequence of the DNA substrate in vitro.
Figure 3. Differential effect of DNA-PKcs on WRN and BLM helicase activities. BLM (3.3 nM,
lanes 3-5) and either DNA-PKcs (16.7 nM, lane 4) or RPA (16.7 nM, lanes 5
and 6) were incubated in standard reaction buffer prior to addition of the
D-loop substrate. Lane 1: A DNA marker, [32P]-INV annealed with
BB. Lane 7: heat-denatured D-loop substrate denoted by a filled triangle.
DNA-PKcs
does not stimulate WRN helicase on non-D-loop DNA substrates
The
ability of DNA-PKcs to stimulate WRN helicase was also tested on several DNA
metabolic intermediates other than D-loops (Figures 5). These included two
forked duplexes with poly-T 15-mer arms, one with a 34-bp duplex region
containing (TTAGGG)4 and one with a 22-bp duplex region lacking
telomeric repeats (Figure 5A). WRN helicase unwinds the 34-bp forked duplex in
the presence of RPA (Figure 5A, lane 5), as reported previously [23]. Under the
same conditions but in the presence of DNA-PKcs, WRN did not unwind this DNA
substrate (Figure 5, lane 3). WRN unwinds the 22-bp forked duplex with similar
efficiency in the absence or presence of DNA-PKcs or RPA (Figure 5A, lanes 8, 9
and 11). Although RPA is thought to increase the processivity of WRN helicase,
the intrinsic processivity of WRN helicase appears to be sufficient for
unwinding the 22-bp forked duplex used in this experiment. Figure 5B shows
that WRN and BLM helicase unwind a Holliday junction DNA substrate, and that
this activity is not stimulated by DNA-PKcs. These results indicate that
DNA-PKcs stimulates the processivity of WRN helicase on the D-loop substrate
but not on other DNA substrates examined in this study. Because D-loops may be
enriched in telomeric regions in vivo, this is consistent with the
proposed roles of WRN and DNA-PK specifically in telomere length maintenance.
Telomeric DNA can exist in a closed D-loop form or an open
form, with the open form more likely to occur during DNA replication or in
response to DNA damage. Therefore,the ability of DNA-PKcs to
stimulate WRN helicase was also tested on a telomeric DNA substrate that
resembles the telomere in an open conformation (Figure 5C). For this purpose,
a DNA substrate was prepared containing a telomericduplex DNA upstream of G-tail [24]. Note that the polarity of WRN helicase is 3'-5',
allowing it to unwind duplexes with a G-tail, but not duplexes with a 5'-ssDNA
tail. The DNA substrate used in these experiments includes both a G-tailed
duplex as depicted in Figure 5C and a second species, which is likely to be a
bi-molecular G-quadruplexstructure formed by annealing of the ssDNA
tails of two G-tailed duplexes. The latter structure has a slower
electrophoretic mobility than the G-tailed duplex (Figure 5C, lane 1),and
it is destabilized by WRN (Figure 5C, lanes 2-5) or boiling(Figure 5C, lane 6).
WRN
exonuclease degrades the open telomeric DNA substrate starting at the3'-OH
blunt end, and WRN helicase unwinds and releases the shortened strand from theG-tailed duplex (Figure 5C, lane 2). DNA-PKcs did not stimulate WRN
helicase on this DNA substrate (Figure 5C, lanes 2-5). The results suggest
that DNA-PKcs stimulates WRN helicase on a telomeric D-loop substrate, but not
on a G-tailed DNA duplex, an open form of a telomeric D-loop.
Figure 4. Effect of DNA-PKcs on WRN helicase activity on
telomeric and non-telomeric D-loops. WRN wild type (WT) (3.3 nM,
lanes 5, 6, 12, and 13) or WRN (E84A) (3.3 nM, lanes 3, 4, 10, and 11)
was preincubated with DNA-PKcs (16.7 nM, lanes 4, 6, 11, and 13).
A telomeric (lanes 2-6) or a non-telomeric D-loop substrate (lanes 9-13)
was added to the reaction. Lanes 1 and 8: A DNA ladder marker. Lanes 7
and 14: heat-denatured telomeric and non-telomeric D-loop substrates,
respectively, denoted by filled triangles.
Figure 5. DNA-PKcs
fails to alter WRN helicase activity on forked duplex, Holliday junction
and G-tailed telomeric DNA substrates. DNA helicase
assays were carried out in the presence of the indicated proteins and DNA
substrates. (A) WRN (1 nM, lanes 2, 3, 5, 8, 9, and 11) and either
DNA-PKcs (5 nM, lanes 3, 4, 9, and 10) or RPA (5 nM, lanes 5, 6, 11, and
12) were incubated in standard reaction buffer prior to addition of a 34 bp
forked duplex (0.5 nM, lanes 1-6) or a 22 bp forked duplex (0.5 nM, lanes
7-12). (B) WRN (4 nM, lanes 2-5) or BLM (2.5 nM, lanes 8-11), and
DNA-PKcs (4 nM, lane 3; 8 nM, lane 4; 20 nM, lanes 5; 2.5 nM, lane 9; 5 nM,
lane 10; 12.5 nM, lane 11) were incubated with in HJ reaction buffer prior
to addition of Holliday junction (0.5 nM, lanes 1-11). Lane 6: DNA-PKcs
(20 nM) alone.
Lane 12: heat-denatured Holliday junction denoted with filled triangles. (C)
G-tailed duplex (0.5 nM, lanes 1-5 and 7) was incubated with WRN (7.5 nM,
lane 2-5) and DNA-PKcs (6.25 nM, lane 3; 12.5 nM, lane 4; 25 nM, lanes 5
and 7) in standard reaction buffer. Lane 6: heat-denatured G-tailed duplex
denoted by a filled triangle.
Protection
of G- tail by WRN helicase activity
The above studies suggest that DNA-PKcs
stimulates telomere unwinding by WRN in vitro, but do not address
whether this interaction is important in vivo. Nevertheless, previous
studies are consistent with this possibility. In particular, telomere length decreases
more quickly in Terc-/-/DNA-PKcs-/- mice than in Terc-/-
mice [25], and Terc-/-/WRN-/- but not WRN-/-
mice have a telomere dysfunction [26]. Thus,
experiments were performed to test whether the interaction between WRN and
DNA-PKcs is important for telomere length maintenance in vivo (Figure 6). For this purpose, a G-tail telomere hybridization protection assay (HPA)
was performed with DNA purified from U-2 OS cells, which
are telomerase negative. The G-tail telomere HPA assay, shown schematically in
Figure 6A, accurately measures telomere G-tail length. The G-tail telomere HPA
assay was first performed using U-2 OS cells which stably express an shRNA
targeted to WRN or control U-2 OS cells which stably express a scrambled shRNA
with no significanthomology to known human genes (Figure 6B). The
results show that G-tail length is significantly shorter in WRN knockdown
cells. The effect of DNA-PKcs on the G-tail length was examined using the
cells transfected with an siRNA targeted to DNA-PKcs (Figure 6C). G-tail
length was also slightly shorter in DNA-PKcs knockdown cells, compared to cells
transfected with control siRNA. Overexpression of N-terminally EYFP-tagged WRN
(E84A), an exonuclease dead mutant, in the DNA-PKcs knockdown cells reversed
the G-tail shortening. This suggests that endogenous WRN exonuclease is
responsible for a part of this outcome of the shortening in the absence of
DNA-PKcs, and an excess amount of WRN (E84A) prevents the exonuclease from
attacking the G-tail and exhibit unwinding activity. However, a similar result
was obtained by overexpression of N-terminally EYFP-tagged WRN wild type in the
DNA-PKcs knock down cells. There may be a mechanism to support an access of an
exonuclease domain of WRN (E84A) but not WRN wild type to the G-tail in cells
(Figure 6C), because the domain (1-239 amino acids) is important to regulate
its binding to forked duplex, which is resemble a part of D-loop substrate [27].
Figure 6. Quantification of telomere G-tail length by hybridization
protection assay in DNA-PKcs knockdown U-2 OS cells. (A) A
schematic of the HPA for telomere G-tail. Non-denatured genomic DNA was
incubated with acridinium ester (AE)-labeled 29-mer telomere HPA probe.
The AE of unhybridized and mis-hybridized probes was hydrolyzed, and
chemilumines-cence from AE of hybridized probes was measured. (B
and C) G-tail length of cells expressing an shRNA control or an
shRNA against WRN was examined in panel B. G-tail length of cells
transfected with siRNA against control (left), siRNA against DNA-PKcs
(middle left), siRNA against DNA-PKcs with pEYFP-WRN (middle right),
or siRNA against DNA-PKcs with pEYFP-WRN (E84A) (right) was examined in
panel C. The G-tail length in the control cells was represented as
100%. Data are represented as mean +/- standard errors of two independent
experiments.
Discussion
This
study demonstrates that DNA-PKcs stimulates the apparent processivity of WRN
helicase but not WRN exonuclease on telomeric and non-telomeric D-loop
substrates in vitro and that overexpression of WRN helicase reverses
telomere G-tail shortening in vivo caused by knockdown of DNA-PKcs in U-2
OS cells. Based on these results, we propose a model for the role of WRN and
DNA-PKcs in D-loop unwinding (Figure 7). The key points of the model are as
follows: 1) In the absence of DNA-PKcs and WRN exonuclease, WRN helicase
dissociates from DNA prior to release of a full-length invading strand,
resulting in reannealing of the unwound region; 2) when WRN exonuclease
degrades the 3' tail of the invading strand, WRN helicase releases
the shortened invading strand, even in the absence of DNA-PKcs; 3) DNA-PKcs
stimulates WRN processivity, so that exonuclease-deficient WRN or WRN is able
to release an intact or nearly intact invading strand from the D-loop,
respectively. This mechanism would protect telomeric DNA 3'-ends, prevent
telomere shortening, and potentially avoid p53-p21-dependentreplicative
senescence.
The results also indicate that DNA-PKcs stimulates
WRN-catalyzed unwinding of non-telomeric D-loop, implying that WRN and DNA-PKcs
could cooperate to unwind recombination-associated D-loops in genomic regions
other than the telomere. This is consistent
with a possible role of WRN in processing D-loop
intermediates in homologous recombination, which is supported by several in
vitro studies [8,28].
Previous
studies also show that POT1 and RPA, WRN and BLM interacting proteins,
stimulate WRN and BLM-catalyzed unwinding of telomeric D-loop substrates in
vitro [13]. However,
the mechanism(s) of this stimulation may differ from the mechanism by which
DNA-PKcs stimulates WRN helicase. POT1 and RPA are ssDNA binding proteins.
They stabilize ssDNA and prevent ssDNA reannealing, rather than preventing WRN
dissociation from the substrate through their interaction with WRN. Unlike
POT1 and RPA, DNA-PKcs has a low affinity for ssDNA, but high affinity for junctions
between ssDNA and dsDNA [29]. Thus,
DNA-PKcs might bind to the ssDNA/dsDNA junctions of D-loops that have been
partially melted by WRN and prevent ssDNA reannealing. Direct interaction
between WRN and DNA-PKcs was demonstrated [21]. It is
also possible that DNA-PKcs prevents WRN from dissociating from the DNA
substrate, and that this interaction stimulates the processivity of WRN
helicase. Recently, it was reported that deacetylation of histone H3 lysine 9
by SIRT6 is required for the stable association of both
WRN and DNA-PKcs with telomeric chromatin, suggesting the possibility of a role
for SIRT6 to control the interaction between WRN and DNA-PKcs at telomeres [30,31].
Additional studies are needed to determine whether and how Ku70/80 influences
the interaction between DNA-PKcs and WRN, especially because Ku70/80 binds
tightly to WRN and stimulates its exonuclease activity [32].
Figure 7. A model for
protection of G-tails by DNA-PKcs. See text for
detailed description of the model.
Mouse model studies indicate that both
DNA-PKcs deficiency and WRN deficiency synergize with telomerase loss and
shortened telomeres to accelerate the onset of aging related phenotypes. Mice
deficient in both WRN and telomerase recapitulate most of the premature aging
WS phenotypes in the later generation cohorts that experienced telomere
shortening [26,33].
Similarly, mice rendered doubly deficient in DNA-PKcs and telomerase exhibited
accelerated aging-related degenerative phenotypes including tissue atrophy,
compared to singly null mice, and this was further exacerbated in later
generations [34]. The loss
of either WRN or DNA-PKcs in telomerase deficient mice was associated with
accelerated telomere shortening and chromosome fusions [26,34].
Preservation of the telomeric tail is essential for preventing telomere
dysfunction and chromosome fusions [35], and our
biochemical data suggest that WRN and DNA-PKcs cooperate to prevent telomere
tail shortening during processing at telomeric ends in telomerase deficient
cells.
Methods
Cells.
U-2 OS cells stably transfected with a vector
expressing an shRNA plasmid against control or WRN were grown in monolayer
culture in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) supplemented
with 10% fetal bovine serum, 0.2 mg/ml hygromycin B (Invitrogen), 50 μg/mlstreptomycin, and 50 U/ml penicillin as previously reported [36]. The U-2 OS
cells stably expressing untargeted shRNA was transfected with siRNA against
DNA-PKcs (sc-35200, Santa Cruz Biotechnology) or control siRNA (sc-37007, Santa
Cruz Biotechnology), with or without pEYFP-WRN or pEYFP-WRN (E84A) vector,
using lipofectamine 2000 Reagent (Invitrogen). Cells were incubated for 2 days
and harvested.
Plasmids.
DNA fragment encoding wild type WRN and WRN (E84A)
was excised from pBK-WRN and pBK-WRN (E84A) with SalI and SspI,
and ligated into the SalI and SmaI site of pEYFP-C1 vector
(Clontech) to produce pEYFP-WRN and pEYFP-WRN (E84A) vectors, respectively.
Proteins.
His-tagged WRN wild type (WT), WRN (E84A) and human Ku 70/86 were overexpressed
in and purified from Sf9 insect cells using a baculovirus expression system as
previously described [37,38]. Recombinant human RPA was purified from E. coli BL21(DE3)
pLysS transformed with p11d-tRPA as described previously [39]. DNA-PKcs was purified from placenta as described
previously [40]. His-tagged BLM was prepared from yeast strain
JEL-1 [41].
G-tail telomere HPA assay.
The G-tail length was quantified by the HPA using
genomic DNA as described previously[44]. Signal
intensity for each sample was normalized by DNA concentration using NANO drop.
DNA substrate.
BB, BT and 5'-radiolabeled INV were annealed to form a telomeric D-loop [12]. BBmx, BT
and 5'-radiolabeled INVmx were annealed to form a non-telomeric D-loop [13].
5'-Radiolabeled and unlabeled 37-mer oligonucleotides were annealed to form a
22-bp forked duplex [42], an
oligonucleotide 6 and a 5'-radiorabeled oligonucleotide 5 were annealed to form
a 34-bp forked duplex [23], and X12-2,
X12-3, X12-4 and 5'-radiolabeled X12-1 were annealed to form a Holliday
junction [43]. The
G-tailed substrate consisting of a 36-bp duplex with 14-bp of unique sequence
followed by 22-bp of (TTAGGG)3TTAG sequence and a 20 nt 3'-ssDNA
tail of the sequence GG(TTAGGG)3 was constructed as previously
described [24]. Briefly
the substrate was formed by annealing the 5'-end labeled Tel Tail duplex
oligonucleotide [24] into a
hairpin to promote correct alignment of the telomeric repeats. An annealing
reaction was at 95˚C for 5 min, cooled stepwise (1.2 C˚/min) to
60˚C, incubated for 1 hr, and then cooled stepwise (1.2 C˚/min) to
25˚C. The hairpin was digested with EcoRV (New England BioLabs) to
generate a blunt end, and the substrate was purified by PAGE.
Helicase
and exonuclease assays.
D-loop unwinding reactions were performed as
described previously [12]. Briefly, the
indicated amount of WRN or BLM was preincubated with DNA-PKcs, RPA or Ku on ice
prior to addition of DNA. Assays contained 0.5 nM [32P] 5' end-labeled D-loop substrate in 30 μl of standard reaction
buffer [40 mM Tris-HCl (pH 8.0), 4 mM MgCl2, 5 mM DTT, 0.1 mg/ml BSA
and 2 mM ATP]. Aliquots of 20 and 5 μl were electrophoresed in non-denaturing
8% polyacrylamide gels containing 0.1% SDS and 14% denaturing gels,
respectively. Reaction products were quantified using a PhosphorImager and
ImageQuantsoftware (Molecular Dynamics). Reactions using forked
duplex and G-tailed telomere substrates were performed in 20 μl standard
reaction buffer [23,24]. Reactions
using Holliday junction substrates were performed in 20 μl HJ reaction buffer
[40 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 5 mM DTT, 0.1 mg/ml BSA and 2
mM ATP] [43].
Acknowledgments
We thank Dr. Ian D. Hickson (University of Oxford) for
His-tagged BLM, Dr. Marc S. Wold (University of Iowa) for p11d-tRPA vector, Dr.
Junko Oshima (University of Washington) for pBK-WRN and pBK-WRN (E84A), and
Drs. Yie Liu, Chandrika Canugovi, and Avik Ghosh for their technical advice and
critical reading of this manuscript. R.K. was supported by a Research
Fellowship of Japan Society For the Promotion of Science (Japan). This work
was supported in part by the Intramural Research Program of the NIH, National
Institute on Aging, as well as NIH grant CA84442 (to D.A.R), grant ES0515052
(P.L.O.) and Grant-in-Aid for Scientific Research No. 20014015 on priority
areas from the Ministry of Education, Culture, Sports, Science and Technology
of Japan (to H.T).
Conflicts of Interest
The authors of this manuscript have no conflict of
interests to declare.
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