CDK1 differentially regulates G-overhang generation at leading- and lagging-strand telomeres in telomerase-negative cells in G2 phase.
ABSTRACT Human telomeres contain single-stranded 3' G-overhangs that function in telomere end protection and telomerase action. Previously we have demonstrated that multiple steps involving C-strand end resection, telomerase elongation and C-strand fill-in contribute to G-overhang generation in telomerase-positive cancer cells. However, how G-overhangs are generated in telomerase-negative human somatic cells is unknown. Here, we report that C-strand fill-in is present at lagging-strand telomeres in telomerase-negative human cells but not at leading-strand telomeres, suggesting that C-strand fill-in is independent of telomerase extension of G-strand. We further show that while cyclin-dependent kinase 1 (CDK1) positively regulates C-strand fill-in, CDK1 unlikely regulates G-overhang generation at leading-strand telomeres. In addition, DNA polymerase α (Polα) association with telomeres is not altered upon CDK1 inhibition, suggesting that CDK1 does not control the loading of Polα to telomeres during fill-in. In summary, our results reveal that G-overhang generation at leading- and lagging-strand telomeres are regulated by distinct mechanisms in human cells.
[show abstract] [hide abstract]
ABSTRACT: The natural ends of linear chromosomes require unique genetic and structural adaptations to facilitate the protection of genetic material. This is achieved by the sequestration of the telomeric sequence into a protective nucleoprotein cap that masks the ends from constitutive exposure to the DNA damage response machinery. When telomeres are unmasked, genome instability arises. Balancing capping requirements with telomere replication and the enzymatic processing steps that are obligatory for telomere function is a complex problem. Telomeric proteins and their interacting factors create an environment at chromosome ends that inhibits DNA repair; however, the repair machinery is essential for proper telomere function.Nature Reviews Molecular Cell Biology 03/2010; 11(3):171-81. · 39.12 Impact Factor
Article: POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end.[show abstract] [hide abstract]
ABSTRACT: The hallmarks of telomere dysfunction in mammals are reduced telomeric 3' overhangs, telomere fusions, and cell cycle arrest due to a DNA damage response. Here, we report on the phenotypes of RNAi-mediated inhibition of POT1, the single-stranded telomeric DNA-binding protein. A 10-fold reduction in POT1 protein in tumor cells induced neither telomere fusions nor cell cycle arrest. However, the 3' overhang DNA was reduced and all telomeres elicited a transient DNA damage response in G1, indicating that extensive telomere damage can occur without cell cycle arrest or telomere fusions. RNAi to POT1 also revealed its role in generating the correct sequence at chromosome ends. The recessed 5' end of the telomere, which normally ends on the sequence ATC-5', was changed to a random position within the AATCCC repeat. Thus, POT1 determines the structure of the 3' and 5' ends of human chromosomes, and its inhibition generates a novel combination of telomere dysfunction phenotypes in which chromosome ends behave transiently as sites of DNA damage, yet remain protected from nonhomologous end-joining.The EMBO Journal 08/2005; 24(14):2667-78. · 9.20 Impact Factor
[show abstract] [hide abstract]
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.Aging 05/2010; 2(5):274-84. · 5.13 Impact Factor
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Cell Cycle 11:16, 3079-3086; August 15, 2012; © 2012 Landes Bioscience
CDK1 differentially regulates G-overhang
generation at leading- and lagging-strand
telomeres in telomerase-negative
cells in G2 phase
*Correspondence to: Weihang Chai; Email: firstname.lastname@example.org
Submitted: 06/13/12; Revised: 07/06/12; Accepted: 07/12/12
Telomeres protect genome stability and integrity by preventing
chromosome ends from inappropriate degradation, fusions and
recombination.1 Human telomere consists of many kilobases
of (TTAGGG)n tandem repeats and terminates with a single-
stranded 3'-overhang at the G-rich strand termed G-overhang.
The importance of G-overhang is multifaceted. First, G-overhang
serves as the binding site for telomerase.2,3 Lack of G-overhang
prevents telomerase binding to telomeres. Second, G-overhang is
essential for the formation of the unique telomere nucleoprotein
structure termed t-loop, which “caps” the chromosome end and
protects the chromosome end from degradation and inappro-
priate DNA repair activities.4 Failure of maintaining the t-loop
structure leads to chromosome end-to-end fusions.5 In addition,
the mechanism for G-overhang generation contributes to telo-
mere shortening. It has been shown that the length of G-overhang
is influenced by a number telomere binding factors,6-13 suggesting
that telomere binding factors regulate G-overhang generation.
Despite the importance of G-overhang in telomere maintenance,
the mechanism underlying G-overhang generation is largely
During DNA replication, the TTAGGG (G-rich) strand
serves as the template for discontinuous lagging-strand synthe-
sis, while the CCCTAA (C-rich) strand serves as the template
Human telomeres contain single-stranded 3' G-overhangs that function in telomere end protection and telomerase
action. previously we have demonstrated that multiple steps involving C-strand end resection, telomerase elongation
and C-strand fill-in contribute to G-overhang generation in telomerase-positive cancer cells. However, how G-overhangs
are generated in telomerase-negative human somatic cells is unknown. Here, we report that C-strand fill-in is present
at lagging-strand telomeres in telomerase-negative human cells but not at leading-strand telomeres, suggesting that
C-strand fill-in is independent of telomerase extension of G-strand. We further show that while cyclin-dependent kinase 1
(CDK1) positively regulates C-strand fill-in, CDK1 unlikely regulates G-overhang generation at leading-strand telomeres.
In addition, DNA polymerase α (polα) association with telomeres is not altered upon CDK1 inhibition, suggesting that
CDK1 does not control the loading of polα to telomeres during fill-in. In summary, our results reveal that G-overhang
generation at leading- and lagging-strand telomeres are regulated by distinct mechanisms in human cells.
Xueyu Dai, Chenhui Huang and Weihang Chai*
School of Molecular Biosciences; Washington State University; Spokane, WA USA
Keywords: telomere, CDK1, G-overhang, C-strand fill-in, telomerase
for continuous leading-strand synthesis. At least four molecular
events are thought to contribute to G-overhang generation. First,
the removal of the final RNA primer and/or the failure to posi-
tion the final RNA primer at the very end of the chromosome
immediately after replication creates an intermediate overhang at
the lagging-strand telomere. Second, the 5' end of C-rich strand
of the initially blunt-ended leading-strand daughter telomere
is resected by nucleases/helicases such as mammalian Apollo,
ExoI14 as well as undefined nucleases, generating a G-overhang
that is essential for telomerase binding. It is currently unknown
whether end resection also takes place at the lagging-strand telo-
mere, although a recent mammalian study suggests so.14 Next,
telomerase extends G-rich strands of both leading- and lagging-
strand telomeres, further lengthening G-overhang.15 Finally, at
least in telomerase-positive cells, a distinct DNA synthesis step
termed C-strand fill-in synthesizes more C-rich repeats in the late
S/G2 phase after the synthesis of genome DNA completes, short-
ening G-overhang length.7,15,16
The C-strand fill-in step is of significant importance to telom-
erase-expressing cells, because it replenishes C-strand length after
telomerase elongates G-strand.16 C-strand fill-in is also present at
yeast and mouse telomeres.17-20 Interestingly, C-strand fill-in in
yeast cells is independent of telomerase expression,18,21-23 suggest-
ing that telomerase extension of telomeres is not a prerequisite for
fill-in. Another possible significance of C-strand fill-in is that,
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understand the mechanism regulating this reconfiguration, we
performed a comprehensive analysis of G-overhangs at leading-
and lagging-strand telomeres in G2 phase. We report here that
C-strand fill-in is present at lagging-strand telomeres in telomer-
ase-negative human cells, suggesting that C-strand fill-in occurs
independent of telomerase. In contrast, C-strand fill-in is absent
at leading-strand telomeres in both telomerase-negative and -pos-
itive cells. Chromatin immunoprecipitation (ChIP) assay reveals
that CDK1 has no effect on the association of DNA Polα to
telomeres at G2 phase, indicating that CDK1 may regulate Polα
activity during C-strand fill-in rather than Polα recruitment to
telomeres. In addition, we also found that while CDK1 positively
regulates C-strand fill-in, it unlikely controls G-overhang genera-
tion at leading telomeres. Our results provide insights into the
molecular mechanism underlying the regulation of G-overhang
generation at human telomeres.
3080 Cell Cycle Volume 11 Issue 16
tested a different telomerase-negative human somatic cell line
BJ/E6/E735 and analyzed G-overhang dynamics during the cell
cycle in BJ/E6/E7. Cells was synchronized at G1/S boundary
using serum starvation/aphidicolin arrest, released into drug-free
media and collected at indicated time during the cell cycle (Fig.
1). FACS analysis shows that nearly 80% cells were synchronized
(Fig. 1A). Genome DNA was isolated, and the overhang protec-
tion assay35 was used to measure G-overhang length. The length
of G-overhangs in BJ/E6/E7 increased during S phase, reached
the longest in late S/G2 phase (6 h to 8 h) and then shortened
in G2 phase (Fig. 1B and C), largely similar to the G-overhang
dynamics in IMR90.7 Thus, our results establish that C-strand
fill-in occurs independent of telomerase, arguing against the con-
cept that C-strand fill-in is exclusively for replenishing C-strand
length after telomerase extends G-strand.
C-strand fill-in occurs at lagging-strand telomeres in telom-
erase-negative cells. To determine whether G-overhangs are
equally shortened in late S/G2 phase at leading- and lagging-strand
telomeres in telomerase-negative cells, we measured G-overhangs
dynamics of separated leading and lagging telomeres from late S
to the completion of G2/M phase. BJ/E6/E7 cells were synchro-
nized at G1/S boundary and then released into S phase. BrdU
was added into media following release, and cells were collected
from late S phase to the next G1 phase (Fig. 2B). Leading- and
lagging-strand telomeres were separated and purified by CsCl
ultracentrifugation (Fig. 2A; Fig. S1).7,36 Non-denaturing in-gel
hybridization was used to determine the G-overhang abundance.
We found that in BJ/E6/E7, G-overhangs of lagging-strand
telomeres shortened during G2 phase (Fig. 2C and D, DMSO-
treated samples), suggesting that C-strand fill-in takes place at
lagging telomeres in telomerase-negative cells.
CDK1 positively regulates C-strand fill-in at lagging-strand
telomeres. Our previous results indicate that CDK1 plays a role
in G-overhang generation.7 Because only one CDK1 inhibi-
tor, purvalanol A (PA), was used in previous study, we there-
fore first determined the specificity of CDK1 inhibition using
other highly selective CDK1 inhibitors CGP74514A (CGP)
and RO3306,37-40 together with PA. HeLa was synchronized at
G1/S boundary using double-thymidine block and then released
into S phase. Since CDK1 activity is low in S phase, increases in
late S/G2 and peaks at M phase, whereas CDK2 activity peaks
in late G1/S phase and mainly controls G1 to S phase transi-
tion,41 CDK1 inhibitors were added to media in late S/G2 phase
(6.5 hr after release) to avoid possible nonspecific inhibition of
other CDKs during S or G1 phase. In addition, the concentra-
tions of inhibitors were carefully determined, so that minimal
amounts of inhibitors were used to achieve CDK1 inhibition
(Fig. S2). Under our experimental conditions, 8 μM PA, 6 μM
CGP and 9 μM RO3306 were sufficient to inhibit CDK1 activ-
ity (Fig. S2). Cells were then collected at different time points,
and the telomere overhang protection assay was used to deter-
mine the mean length of G-overhangs (Fig. S3). In DMSO-
treated control cells, G-overhang length shortened from late S/
G2 to the next G1 phase, whereas treatments with PA, CGP and
RO3306 attenuated such G-overhang shortening (Fig. S3C). We
also treated HeLa with roscovitine, a commonly used CDK1/2
since the final Okazaki fragment may not be placed at the very
end of lagging-strand telomere,24 it will leave the daughter strand
significantly shorter and telomere will undergo rapid shortening
without C-strand fill-in.
G-overhang length at human telomeres undergoes cell cycle-
regulated dynamics, with G-overhang length transiently increas-
ing during S phase and then reducing during late S/G2 phase
in telomerase-positive cells.7 Inhibition of the activity of CDK1
results in the retention of long G-overhangs at global telo-
meres in the G2/M phase, suggesting that CDK1 may regulate
G-overhang generation.7 CDKs are a series of kinases controlling
the precise progression of the cell cycle. CDK consists of a cata-
lytic kinase subunit and a positive regulatory subunit (cyclin).25
It is generally thought that CDK4/6 with D cyclins control G1
phase progression; CDK2/cyclins E and A control S phase pro-
gression; and CDK1/cyclins A and B trigger G2/M progression.26
In human cells, CDK1 regulates the resolution of sister telomeres
through phosphorylating TRF1.27 In budding yeast, which con-
tains only one CDK, cdc28, Cdc28 is required for the genera-
tion of G-overhangs by controlling end resection28-30 as well as
by regulating the recruitment of telomerase to telomeres.31,32 The
role of CDK1 in regulating telomere maintenance in human cells
is largely elusive.
In the G2 phase of the cell cycle, a variety of proteins, includ-
ing the ones involved in general DNA replication, recombination
and repair, become associated with telomere DNA,33,34 suggest-
ing that telomeres are under active reconfiguration, and that
critical events take place at telomeres in the G2 phase. To better
Global G-overhangs are lengthened during S phase and short-
ened in G2 phase in telomerase-negative human cells. In a
previous study we analyzed G-overhang dynamics in telomer-
ase-negative human somatic cell line IMR90, and found that
G-overhangs were lengthened in S phase and then shortened
in G2 phase.7 This was a surprising finding, because C-strand
fill-in was viewed as a means for replenishing C-strand length
after telomerase extension of G-strand. To validate the G2 phase-
specific G-overhang shortening in the absence of telomerase, we
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leading telomeres from late S to G2 phase in BJ/E6/E7,
and this increase was statistically significant (Fig. 3B,
DMSO-treated samples). Since telomerase activity is
absent in BJ/E6/E7 cells, this increase suggested that
in addition to the initial end resection in S phase, an
additional end resection step takes place at C-strand of
the leading telomere in late S/G2 phase. This resection
might be due to the continuation of the initial resection
at a portion of leading telomeres or a distinct second
resection independent of the initial end resection. The
nature of C-strand end resection, both the initial and
the second ones, remains to be elucidated.
We then analyzed whether CDK1 regulated
G-overhang generation at leading-strand telomeres in
G2 phase. Treatment with PA did not significantly alter
G-overhang length at leading telomeres in either BJ/
E6/E7 or HeLa (Fig. 3B and D), indicating that CDK1
does not play a regulatory role in G-overhang genera-
tion at leading telomeres.
The recruitment of Polα to telomeres is not regu-
lated by CDK1. Polα activity is required for C-strand fill-in.7
We then determined whether CDK1 regulates the recruitment
of Polα to telomeres during the late S to G2 phase using chroma-
tin immunoprecipitation (ChIP) assay (Fig. 4). HeLa cells were
synchronized, released into S phase and collected at indicated
time (Fig. 4A). PA was added at middle to late S phase (5.5 h
after release). ChIP was performed with the anti-Polα antibody,
and the precipitated DNA was denatured, loaded on slot blot and
www.landesbioscience.com Cell Cycle 3081
hybridized to telomere probe. Consistent with previous report,34
Polα re-associates with telomeres at G2 phase in DMSO control
cells, likely to complete C-strand fill-in at this time. Interestingly,
CDK1 inhibition with PA showed no significant change in Polα
telomeric association (Fig. 4C), indicating that CDK1 did not
regulate the recruitment of Polα to telomeres. It is possible that
CDK1 may control C-strand fill-in by regulating the RNA prim-
ing and/or synthesis activity of Polα.
inhibitor,42 at late S phase (6 h after release), and found
that roscovitine also blocked G-overhang shortening in
G2 (Fig. S4). We previously showed that the attenu-
ation of G-overhang shortening was not due to cell
cycle arrest.7 Collectively, our results demonstrate that
CDK1 regulates C-strand fill-in in G2 phase.
To determine whether CDK1 regulates G-overhang
generation at both daughter telomeres, synchronized
BJ/E6/E7 cells were released into BrdU-containing
media, and PA was added into the media at late S phase
to inhibit CDK1 activity. Cells were collected from late
S phase to the next G1 phase (Fig. 2B). Addition of PA
abolished G-overhang shortening at lagging-strand
telomeres in BJ/E6/E7 (Fig. 2C and D), suggesting
that CDK1 positively regulates the fill-in step at lag-
C-strand fill-in is absent at leading-strand telo-
meres, and CDK1 unlikely regulates G-overhang
generation at leading telomeres. When G-overhang
dynamics of leading telomeres was measured, we
found that G-overhang length of leading telomeres did
not decrease from S to G2/M phase in BJ/E6/E7 cells
(Fig. 3A and B, DMSO-treated samples). Similarly,
leading G-overhangs were not shortened in HeLa cells
(Fig. 3C and D, DMSO-treated samples). Collectively,
our results suggest that C-strand fill-in is absent at lead-
We noticed that G-overhang abundance increased at
Figure 1. G-overhang length dynamics of telomerase-negative BJ/e6/e7 cells
during the cell cycle. (A) FACS analysis of DNA content in synchronized BJ/e6/e7
cells (pD39). Cells were collected from 0 h to 12 h after release from G1/S boundary.
(B) G-overhang length measurement of genomic DNA using the overhang protec-
tion assay. (C) Quantitation of mean overhang lengths. two-tailed t-test was used
to calculate statistical significance. Results were from two independent experi-
ments. error bars: s.e.m.
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Figure 2. G-overhang dynamics at lagging telomeres in BJ/e6/e7 with or without CDK1 inhibition. (A) Scheme of separation of leading and lagging
daughter telomeres.36 (B) FACS analysis of DNA content in synchronized BJ/e6/e7 cells (pD37). DMSo and 8 μM pA was added at 6 hr after release. Cells
were collected at 7 h, 9.5 h, 12 h and 14.5 h (next G1) after release. (C) G-overhang abundance at lagging-strand telomeres measured by non-denatur-
ing in-gel hybridization assay. (D) Quantitation of G-overhang abundance. two-tailed t-test was used to calculate statistical significance. Results were
from at least three independent experiments. error bars: s.e.m.
3082 Cell Cycle Volume 11 Issue 16
HeLa cells. However, C-strand fill-in is lacking at leading telo-
meres in HeLa (Fig. 3D), arguing against the notion that fill-in
is dependent on telomerase extension of G-strand. So why does
C-strand fill-in exist in telomerase-negative cells? We consider
that the fill-in step is most likely necessary due to two reasons.
First, the final RNA primer may not be positioned at the very end
of a telomere, as suggested by a recent study.24 In addition, during
the processing of the final Okazaki fragment, a significant por-
tion of C-strand DNA can be removed from the 5' end of lagging-
strand telomere.14,43 If no mechanism fills in the gap, the lagging
daughter strand would be rapidly shortened. C-strand fill-in,
perhaps with a mechanism distinct from conventional lagging-
strand synthesis,15 can attenuate such rapid telomere shortening
in each replication. At the leading-strand telomere, such a prob-
lem does not exist, and end resection produces a much shorter
G-overhang (less than 1/2 of the length of lagging overhang)
(Fig. 3E and F).36 This short G-overhang may not trigger a signal
for C-strand fill-in. Given the importance of C-strand fill-in in
telomere shortening, understanding the mechanism regulating
C-strand fill-in will aid in developing novel therapies for prevent-
ing accelerated aging caused by rapid telomere shortening.
We used both telomerase-negative and -positive cells to determine
the molecular mechanism regulating G-overhang generation at
telomeres replicated by leading- and lagging-strand syntheses.
Our results show that in telomerase-negative cells, G-overhang
shortening in G2 phase exists at global telomeres (Fig. 1C).
Further study reveals that such G2-specific G-overhang shorten-
ing is caused by C-strand fill-in at lagging telomeres, and CDK1
positively regulates the fill-in step (Fig. 2D). In addition, our
results demonstrate that C-strand fill-in is absent at leading-
strand telomeres, and CDK1 plays a minimal role in regulating
G-overhang generation at leading telomeres (Fig. 3B and D). Our
results firmly establish that G-overhang generation at leading- and
lagging-strand telomeres are regulated by distinct mechanisms.
Two pieces of evidence support our conclusion that C-strand
fill-in is independent of telomerase expression in human cells.
First, G-overhang shortens in G2 phase in telomerase-negative
cells (Fig. 2D).7 Second, if C-strand fill-in is exclusively used for
replenishing C-strand after telomerase extends G-strand, a fill-in
step should be observed at leading telomeres in telomerase-positive
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This paradox highlights the complexity of end resection and telo-
mere protection in human cells. Since the blunt-ended leading
daughter telomeres resemble DSBs, it will be interesting to know
whether enzymes involved in resecting double-strand breaks are
the key players in resecting telomere ends.
Cell culture and synchronization. All cells were cultured at 37°C
under 5% CO2 in DMEM supplemented with 10% fetal bovine
One important step controlling the generation of G-overhang
as well as the rate of telomere shortening is end resection of
C-strand. It will be important to understand the mechanisms
underlying the regulation of end resection, because end resection
needs to be tightly regulated to avoid excessive degradation of
C-strand. In yeast, end resection is limited by the telomere pro-
tection protein complex Cdc13/Stn1/Ten1.44-46 In mammalian
cells, Pot1b appears to play this role.14,47,48 However, depletion
of human Pot1 leads to G-overhang shortening,6,8 contradict-
ing the expectation from protecting C-strand from degradation.
Figure 3. G-overhang dynamics at leading telomeres from BJ/e6/e7 and HeLa with or without CDK1 inhibitor. (A) G-overhang abundance at leading
daughter telomeres from BJ/e6/e7 measured by non-denaturing in-gel hybridization assay. (B) Quantitation of G-overhang abundance at leading telo-
meres from BJ/e6/e7. (C) G-overhang abundance at leading-strand telomeres from HeLa measured by non-denaturing in-gel hybridization assay. (D)
Quantitation of G-overhang abundance at leading telomeres from HeLa. two-tailed t-test was used to calculate statistical significance. Results were
from at least three independent experiments. error bars: s.e.m. (e and F) show that leading overhangs are much shorter than lagging overhangs in BJ/
e6/e7 without CDK1 inhibition (e) and with CDK1 inhibition (F).
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3084 Cell Cycle Volume 11 Issue 16
to leading-strand telomeres due to the high content of BrdU and
can lead to degradation of leading telomeres.49 Thus, the amount
of leading-strand telomeres appeared to be lower than that of lag-
ging telomere in UV-treated samples, whereas the amounts of
leading and lagging telomeres were similar in samples treated
with baking (Fig. S1). Fractions containing leading or lagging
telomeres were combined; the DNA was desalted, concentrated
and used in G-overhang analysis. All procedures were performed
Telomeric G-overhang length measurement. The mean
length of the telomeric G-overhang was measured by overhang
protection assay as described previously.35
denaturing in gel hybridization was performed as described
previously.7 Briefly, genomic DNA or isolated leading and lag-
ging telomeres was treated with or without ExoI at 37°C for 3
h, followed with proteinase K at 55°C for 2 h and was resolved
on 0.8% 0.5 x TBE agarose gel. The gel was then dried at r.t.,
hybridized to telomeric C-rich probe at 37°C for overnight,
washed with washing buffer (0.1XSSC, 0.1% SDS) two times
at 30°C and exposed to PhosphoImager (GE Healthcare). In
the native gel, the signal from the ExoI-treated samples (ExoI +)
represented the background and the signal from untreated sam-
ples (ExoI-) represented the G-overhang signal. The gel was
then denatured in denaturing buffer (0.5 M NaOH, 1.5 M
NaCl), rinsed with H2O, neutralized with neutralization buffer
hybridization assay. Non-
serum (FBS) or cosmic calf serum (Hyclone). Double thymidine
block was used to synchronize HeLa cells as described previ-
ously.7 For synchronization of BJ/E6/E7, cells were serum starved
for 48 h, split into DMEM media containing 20% FBS with 1
μg/mL aphidicolin for 24 h and then released into DMEM-20%
FBS. Cells were then collected at different time points for analyz-
ing DNA contents using a Beckman Coulter EPICS® XL™ flow
cytometer. To inhibit CDK1 activity, the CDK1 inhibitors were
dissolved in DMSO and added at indicated times and concentra-
tions. Cells treated with DMSO were used as the control.
Separation of leading and lagging daughter telomeres.
Leading and lagging daughter telomeres were isolated as
described7 with minor modifications. Briefly, BJ/E6/E7 or HeLa
cells were synchronized at G1/S boundary and released into
media containing 100 μM BrdU. Genomic DNA isolated from
BrdU-labeled cells was digested with three restriction enzymes
HaeIII, RsaI and AluI, mixed with CsCl solution and subjected
to ultracentrifugation at 44,000 rpm for 13 h. After fractions
were collected from bottom (high density) to top (low density),
CsCl density at each fraction was measured by refractometer, and
the amount of telomere DNA in each fraction was determined by
hybridization to C-rich probe using slot-blot. DNA analyzed in
the upper panel in Figure S1 was crosslinked to membrane with
UV treatment, while DNA samples in the lower panel in Figure
S1 were crosslinked to the membrane by baking at 95°C for 2 h.
It has been shown that UV light, but not baking, is damaging
Figure 4. the association of polα to telomeres was not altered upon CDK1 inhibition. (A) FACS analysis of DNA content in synchronized HeLa used for
ChIp. (B) Representative slot-blot results from ChIp of polα. (C) Quantitation of ChIp of polα. two-tailed t-test was used to calculate statistical signifi-
cance. Results were from at least three independent experiments. error bars: s.e.m.
© 2012 Landes Bioscience.
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malian Ctc1-Stn1-Ten1 complex binds to single-
stranded DNA and protects telomeres independently
of the Pot1 pathway. Mol Cell 2009; 36:193-206;
65; PMID:20697207; http://dx.doi.org/10.4161/
www.landesbioscience.com Cell Cycle 3085
Na-Deoxycholate, 1 mM EDTA, 10 mM TRIS-HCl pH8), buf-
fer D (1 mM EDTA, 10 mM TRIS-HCl pH8.0). For each wash,
1 ml of buffer was incubated with beads for 5 min at 4°C. Beads
were then washed again with buffer D for 5 min and eluted with
300 μL buffer (1% SDS, 100 mM NaHCO3) at 55°C for 15 min.
Elutes were reverse crosslinked in 200 mM NaCl at 65°C for 6 h
to overnight, treated with 20 μg of DNase-free RNase A at 37°C
for 30 min, followed by 20 μg protease K treatment at 45°C for
1 h. DNA was then precipitated by ethanol and used for slot-blot
to hybridize to telomeric probe.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
X.D. and C.H. performed experiments and analyzed data. X.D.
and W.C. wrote the paper.
This work was supported by National Institute of Health
R15GM099008 to W.C.
Supplemental materials may be found here:
(0.5 M TRIS-HCl pH7.5, 1.5 M NaCl), rehybridized to telo-
meric C-rich probe at 42°C for overnight, washed and exposed
to PhosphoImager. The signal from the denatured gel repre-
sented the total amount of telomere DNA. The signal from
native gel normalized by signal from denatured gel represented
the G-overhang abundance.
ChIP assay. About 10 million cells were cross-linked with 1%
formaldehyde in PBS for 10 min. Glycine (0.2 mM) was added
to stop crosslinking. Cells were then collected with scraper, resus-
pended in lysis buffer [1% SDS, 10mM EDTA pH8.0, 50 mM
TRIS-HCl pH8.0, 1 mM PMSF, 1 x protease inhibitor (Roche)],
sonicated (10 sec pulse with 1 min intervals on ice, eight times)
and centrifuged at 4°C for 10 min at 20,000 g. Supernatant
was then diluted with dilution buffer (0.01% SDS, 1.1% Triton
X-100, 1.2 mM EDTA, 16.7 mM TRIS-HCl pH8.0, 150 mM
NaCl, 1 mM PMSF, 1 x protease inhibitor) and incubated with
30 μL 50% protein G beads (Roche) at 4°C for 1 h to preclear
the lysate. Precleared lysate was then incubated with antibody at
4°C for overnight. Protein G beads were then added to the mix-
ture and incubated for 1 h at 4°C. After pelletting at 2,000 g for
2 min, beads were washed sequentially with buffer A (0.1% SDS,
1% Triton X-100, 2 mM EDTA, 20 mM TRIS-HCl pH8.0,
150 mM NaCl, 1 mM PMSF, 1 x protease inhibitor), buffer B
(0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM TRIS-HCl
pH8.0, 500 mM NaCl), buffer C (250 mM LiCl, 1% NP-40, 1%
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