The Journal of Cell Biology, Volume 153, Number 2, April 16, 2001 367–380
The Rockefeller University Press, 0021-9525/2001/04/367/14 $5.00
Regulation and Localization of the Bloom Syndrome Protein in Response
to DNA Damage
Oliver Bischof,* Sahn-Ho Kim,* John Irving,
*Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720;
California 94804; and Department of Human Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Nathan A. Ellis,
and Judith Campisi*
Berlex Laboratories, Inc., Richmond,
cessive disorder characterized by a high incidence of
cancer and genomic instability. BLM, the protein defec-
tive in BS, is a RecQ-like helicase, presumed to function
in DNA replication, recombination, or repair. BLM
localizes to promyelocytic leukemia protein (PML)
nuclear bodies and is expressed during late S and G2.
We show, in normal human cells, that the recombination/
repair proteins hRAD51 and replication protein (RP)-A
assembled with BLM into a fraction of PML bodies
during late S/G2. Biochemical experiments suggested
that BLM resides in a nuclear matrix–bound com-
plex in which association with hRAD51 may be di-
rect. DNA-damaging agents that cause double strand
breaks and a G2 delay induced BLM by a p53- and
Bloom syndrome (BS) is an autosomal re- ataxia-telangiectasia mutated independent mechanism.
This induction depended on the G2 delay, because it
failed to occur when G2 was prevented or bypassed. It
coincided with the appearance of foci containing BLM,
PML, hRAD51 and RP-A, which resembled ionizing
radiation-induced foci. After radiation, foci containing
BLM and PML formed at sites of single-stranded DNA
and presumptive repair in normal cells, but not in cells
with defective PML. Our findings suggest that BLM is
part of a dynamic nuclear matrix–based complex that
requires PML and functions during G2 in undamaged
cells and recombinational repair after DNA damage.
matrix • homologous recombination
RECQ helicases • p53 • ATM • nuclear
Several genes have evolved to ensure the integrity and
stability of cellular genomes. Some of these genes are
conserved from bacteria to humans, whereas others are
restricted to eukaryotes or mammals. In mammals, failure
to maintain genomic stability almost inevitably leads to
cancer. At present, we have only a rudimentary under-
standing of the pathways by which genomic stability is
maintained in mammalian cells, particularly human cells.
Recent findings implicate genes related to
in maintaining genomic stability in human
encodes a DNA helicase that acts in homolo-
gous recombination (HR) and suppresses illegitimate re-
combination, particularly during the repair of DNA dou-
ble strand breaks (DSBs; Hanada et al., 1997; Harmon and
throughout evolution. Budding and fission yeast have a
single gene (SGS1, RQH1/RAD12), which participates in
recombination and chromosome segregation (Stewart et
al., 1997; Watt et al., 1997; Davey et al., 1998). Mammalian
-like functions to maintain genomic in-
tegrity, but, in contrast to
-like genes. Five human
(Puranam and Blackshear, 1994; Seki et al., 1994; Ellis et
al., 1995; Yu et al., 1996; Kitao et al., 1998). Each contains
a region with strong homology to seven motifs that specify
helicase activity. Indeed, biochemical assays
showed that RECQL, BLM, and WRN encode proteins
that are 3
DNA helicases (Tada et al., 1996; Gray et
al., 1997; Karow et al., 1997). Despite strong homology in
the helicase domains, human
markedly outside these domains. Moreover, three of these
genes are associated with autosomal recessive disorders
that, despite some similarities, display striking phenotypic
-like genes are prevalent
and yeast, have multiple
-like genes have
-like genes differ
Address correspondence to Judith Campisi, Lawrence Berkeley National
Laboratory, Mailstop 84-171, Berkeley, CA 94720. Tel.: (510) 486-4416.
Fax: (510) 486-4545. E-mail: firstname.lastname@example.org
O. Bischof’s present address is Institute Pasteur, 28 rue de Dr. Roux,
75724 Paris, Cedex 15, France.
Abbreviations used in this paper:
AT, ataxia telangiectasia; ATM, AT
mutated; BS, Bloom syndrome; DSB, double-strand break; DNA-PK,
DNA-dependent protein kinase; GST, glutathione
mologous recombination; HU, hydroxyurea; IR, ionizing radiation; IRIF,
IR-induced foci; LN, labeled nuclei; NB, nuclear body; PARP, poly-ADP
ribose polymerase; PML, promyelocytic leukemia protein; RP, replication
protein; RT, reverse transcriptase; RTS, Rothmund-Thomson syndrome;
SCE, sister chromatid exchange; WS, Werner syndrome.
-transferase; HR, ho-
The Journal of Cell Biology, Volume 153, 2001
The first human
reditary disorder was
syndrome (BS). Individuals with BS suffer from acute symp-
toms, including pre- and postnatal growth retardation, im-
munodeficiency, and male infertility. BS individuals also
have a very high incidence of cancer. Cancer is the primary
cause of death, which generally occurs before the third de-
cade of life. BS cells are hypermutable, showing numerous
chromatid gaps and breaks and many sister chromatid ex-
changes (SCEs) (German, 1993; Ellis and German, 1996;
Watt and Hickson, 1996). Defects in
have also been linked to hereditary disorders: Werner syn-
drome (WS), in the case of
mund-Thomson syndrome (RTS), in the case of
WS and RTS share several features with BS, most notably a
high incidence of cancer (Vennos and James, 1995; Watt and
Hickson, 1996; Martin et al., 1999). In addition, WS and RTS
cells, like BS cells, are hypermutable (Fukuchi et al., 1989;
Miozzo et al., 1998). However, there are marked differences.
WS individuals are asymptomatic before puberty, but there-
after develop a panoply of age-related disorders, including
cardiovascular disease, cataracts, and osteoporosis (Goto,
1997). RTS individuals have distinctive skin and skeletal ab-
normalities (Starr et al., 1985; Vennos and James, 1995).
Moreover, despite being hypermutable, WS and RTS cells
do not show the high rate of SCE characteristic of BS cells.
The high incidence of cancer and genomic instability in
BS, WS, and RTS suggest that the functions of
may overlap. On the other hand, the pheno-
typic differences among BS, WS, and RTS suggest that these
genes have distinct functions. By analogy with
-like genes are presumed to function in DNA
replication, recombination, and/or repair. However, little is
known about the specific processes in which they participate.
expression, in contrast to that of other human
-like genes (Gray et al., 1998; Kitao et al., 1998), is
cell cycle regulated, peaking in late S phase and G2
(Gharibyan and Youssoufian, 1999; Dutertre et al., 2000),
and localizing to nuclear foci containing the promyelocytic
leukemia protein (PML) tumor suppressor (Gharibyan
and Youssoufian, 1999; Ishov et al., 1999; Zhong et al.,
1999). We show that BLM protein and foci are also in-
duced by agents that cause DNA DSBs. This induction
was indirect and due to the G2 delay caused by the agents.
BLM foci also contained the recombination/repair pro-
teins hRAD51 and replication protein (RP)-A and BLM
was a component of the RAD51 ionizing radiation-
induced foci (IRIF), which form in response to ionizing ra-
diation (IR) (Haaf et al., 1995; Maser et al., 1997; Golub et
al., 1998). Moreover, both BLM and PML associated with
sites of putative repair after IR-induced damage. Our re-
sults suggest that BLM is a component of a nuclear ma-
trix–based complex, which resides in the PML bodies and
forms increasingly during G2 in undamaged cells and in
response to potentially recombinogenic DNA damage.
-like gene that was linked to a he-
, the gene defective in Bloom
, and a subset of Roth-
Materials and Methods
Proliferative capacity was assessed by labeling cells for 72 h with [
Ci/ml) and autoradiography to determine the percentage of
radiolabeled nuclei (% LN) (Dimri et al., 1994, 1995). Human WI-38 fetal
lung fibroblasts were used at early passage (
10% LN), as indicated. E6-expressing WI-38 (Dimri et al., 2000) cells
were generated by retroviral transfer of the human papilloma virus E6
gene (Halbert et al., 1992). E. Blakely and D. Chen (Lawrence Berkeley
National Laboratory) provided ataxia telangiectasia (AT)-2SF and
MO59J (DNA-PKcs–deficient) cells, respectively; J. German (Cornell
Medical School, Ithaca, NY) provided BS fibroblasts HG2654; and O.
Pereira-Smith (Baylor College of Medicine, Houston, TX) provided
HT1080 and HCA2 cells. VA-13 and SAOS-2 cells, and BS fibroblasts
GM001492F were from the American Type Culture Collection. Unless
noted otherwise, cells were cultured in Dulbecco’s modified Eagle’s me-
dium containing 10% fetal bovine serum.
70% LN) or senescence
Cell Cycle Measurements
Proliferating cells were made quiescent by culturing in 0.2% serum for 72–
96 h and stimulated to proliferate with 10% serum. To block cells at G1/S,
quiescent cells were stimulated for 10–12 h, given 5 mM hydroxyurea
(HU; Sigma-Aldrich) for 12–16 h in 10% serum, and released by washing
and providing drug-free 10% serum (Lu et al., 1989). Entry into and
through S phase was monitored by a 1-h pulse with [
toradiography, as described above. Cell cycle distributions were assessed
by flow cytometry of propidium iodide–stained nuclei (Pucillo et al., 1990)
using a FACScan™ and Lysis II software (Becton Dickinson). Mitotic in-
dices were determined by fixing cells in 3:1 methanol/acetic acid, staining
with 0.5 mg/ml propidium iodide in PBS, and counting mitotic figures by
fluorescence microscopy. 1,000 nuclei were counted for each point.
H]thymidine and au-
Irradiation and Drug Treatment
Cells were X-irradiated (1.8 Gy/min) in 10% serum using a PANTAK
HF160 generator (Comet Ag), and UV-irradiated (1.6 J/m
ing a UVC GTE G875 bulb. Bleomycin (10 mg/ml in PBS), etoposide (10
mM in dimethylsulfoxide; Calbiochem), hydrogen peroxide (30%), and
caffeine (Sigma-Aldrich) were diluted into 10% serum before use. Cells
were given media containing drug or solvent for 1 h at 37
otherwise, washed, and given drug-free 10% serum.
/s) in PBS us-
C, unless noted
The affinity-purified rabbit anti-BLM antibody has been described (Ishov
et al., 1999; Neff et al., 1999; Zhong et al., 1999). It did not detect an
kD band in Western blots of BS cells (not shown). Rabbit anti-hRAD51
IgG was a gift from Dr. D. Chen (Lawrence Berkeley National Labora-
tory) or purchased from Oncogene Research Products.
and RP-A (Ab1, Ab2) antibodies were from Oncogene Research Prod-
ucts, BrdU antibody was from Boehringer, poly-ADP ribose polymerase
(PARP) antibody (H-250) was from Santa Cruz Biotechnology, Inc., anti-
Ku70 (clone N3H10) was from NeoMarkers, and fluorescent or horserad-
ish peroxidase–conjugated secondary antibodies were from Vector Labo-
ratories or Bio-Rad Laboratories.
Cells in 4-well glass slides were cultured 2–4 d before irradiation or syn-
chronization, fixed and stained as described (Compton et al., 1991), incu-
bated with primary antibody for 2 h, and secondary (fluorescein isothiocy-
anate– or Texas red–labeled) antibody for 1 h. BLM and hRAD51 were
detected using a Fab fragment secondary antibody (Wessel and McClay,
1986; Jackson ImmunoResearch Laboratories). Slides were mounted in
VectaShield containing DAPI (0.4
g/ml; Vector Laboratories) to visual-
ize nuclear DNA and viewed by epifluorescence or a single laser confocal
section. Foci (at least 200 nuclei/data point) were scored at 600
cation. Images were captured with a CCD camera and merged using Can-
Detection of Single Strand DNA after Damage
Cells were grown on slides in 10% serum containing 10
30 h and X-irradiated in BrdU-free medium as described (Raderschall et
al., 1999). Cells were fixed at the indicated times after irradiation (Comp-
ton et al., 1991) and immunostained using mouse anti-BrdU IgG and
FITC-goat anti–mouse IgG (Boehringer), or rabbit anti-BLM IgG and Texas
red goat anti–rabbit IgG. Cells were counterstained with DAPI and
viewed as described above.
g/ml BrdU for
Bischof et al.
BLM and DNA Damage
Total protein lysates were prepared in 2
g protein was separated by 4–15% SDS-PAGE and analyzed by
Western blotting as described (Dimri et al., 1996). Antibodies were de-
tected by chemiluminescence using SuperSignal (PierceChemical Co.).
Signals were quantified by densitometry using ImageQuant software (Mo-
SDS-PAGE sample buffer and
TaqMan Reverse Transcriptase PCR Analyses
Total RNA was prepared using a commercial kit (Promega). One-step re-
verse transcriptase (RT)-PCR was performed using the TaqMan Gold
RT-PCR kit (PerkinElmer) according to the manufacturer’s instructions.
RT-PCR reactions were performed on 40 ng RNA, in triplicate, using an
ABI 7700 Sequence Detection system (Heid et al., 1996). Primer and fluo-
rogenic probes used for
press software (PerkinElmer). The primer/probe sequences were: forward
, probe 5
; forward 5
averaged and normalized to triplicate measurements of 18 S ribosomal
RNA (TaqMan ribosomal RNA control reagent kit) or
1996) mRNA with similar results using 40 pg RNA per reaction. Values
reported are normalized to
were designed using Primer Ex-
, reverse 5
, probe 5
. Triplicate measurements were
(Dimri et al.,
expressed in Sf9 cells infected with recombinant baculoviruses using a
commercial kit (GIBCO BRL). Nuclear lysates (Dignam et al., 1983)
from infected cells were clarified by centrifugation and incubated for 1 h
C with glutathione-Sepharose 6-CL B resin (Amersham Pharma-
cia Biotech). The slurry was transferred to a column and washed with
50 column volumes each of PBS plus 0.2% NP-40 and PBS. Nuclear ly-
sates prepared from SAOS-2 cells were pretreated with DNase I (160
U/ml) for 30 min at 37
C, after which ethidium bromide was added to
g/ml. These nuclear lysates were incubated with glutathione-
Sepharose to which GST or GST-BLM for 1 h at 4
washed with 50 vol each of PBS plus 0.5% NP-40, PBS plus 0.2% NP-
40, and PBS. Proteins were eluted from the resin with 20 mM glu-
tathione, 100 mM Tris, pH 8.0, 150 mM NaCl, 1 mM DTT, and 10%
glycerol, and those remaining after elution were released by boiling in
SDS-PAGE sample buffer. Eluted proteins and proteins released
by boiling (30
g) were analyzed by 4–15% SDS-PAGE and Western
blotting for BLM, hRAD51, poly-ADP-ribose polymerase, and Ku70.
For immunoprecipitation, SAOS-2 nuclear lysates were precleared by
incubating with 5
g rabbit IgG and Dynabeads-protein G (Bio-Rad
Laboratories) for 1 h at 4
C with gentle agitation. The beads were col-
lected and the supernatant was incubated with 5
(Oncogene Research Products) for 16 h at 4
Dynabeads for 1 h at 4
C. The beads were collected, washed exten-
sively with PBS plus 0.2% NP-40, and washed once with PBS. Immune
complexes were released from the beads by boiling in 2
sample buffer, and analyzed by 4–15% SDS-PAGE and Western blot-
ting for BLM and hRAD51.
-transferase (GST) and GST-BLM fusion protein were
C. The resin was
g rabbit anti-RAD51
C, followed by addition of
Nuclear Matrix Preparation
Nuclear matrix was prepared from cells (80% confluent) by either of
two methods as described (Wan et al., 1999). In brief, cells were sus-
pended at 5
10 /ml in 0.3 M sucrose, 3 mM MgCl
mM PIPES, pH 6.9, 100 mM NaCl, proteinase inhibitor mix (Boeh-
ringer), and 10 U/ml prime RNase inhibitor (Promega) (suspension
buffer), permeabilized by addition of 0.5% Triton X-100 for 7 min on
ice, and washed in suspension buffer. Nuclei were collected by centrifu-
gation, digested with DNase I (300 U/ml, 37
extracted by 0.25 M ammonium sulfate followed by 2 M NaCl or two in-
-hydroxysulfosuccinimide acetate (Pierce Chemical
Co.), all in suspension buffer, for 20 min at room temperature. The pel-
let was digested with RNase A (10
min on ice, solubilized by addition of 2% SDS, and analyzed by SDS-
PAGE and Western blotting.
, 1 mM EGTA, 10
C, 30 min), pelleted, and
g/ml in suspension buffer) for 20
BLM Localizes to PML Nuclear Bodies with hRAD51
and RP-A during Late S/G2
BLM is expressed most highly during S phase and G2 and
localizes to nuclear foci in a fraction of asynchronously di-
viding cells (Gharibyan and Youssoufian, 1999; Neff et al.,
1999; Dutertre et al., 2000). These foci were identified as
PML nuclear bodies (NBs) (Ishov et al., 1999; Zhong et
al., 1999). PML is a tumor suppressor that inhibits cell pro-
liferation and promotes apoptosis in many cells (de The et
al., 1991; Mu et al., 1994; Le et al., 1996; Wang et al.,
To follow assembly of BLM into PML NBs and identify
other components in PML/BLM foci, we monitored pro-
tein localization during cell cycle progression by immuno-
fluorescence. Normal human fibroblasts (WI-38) were ar-
rested in G0 (quiescence) by serum deprivation and then
stimulated by 10% serum to progress synchronously
through G1 and S phase. Alternatively, we arrested cells at
the G1/S boundary by stimulating G0 cells in the presence
of HU; after removing HU, cells progressed synchro-
nously through S, G2, M, and into the next cell cycle (Lu et
al., 1989). We followed the fraction of cells in S by a 1-h
pulse with [
H]thymidine and autoradiography (% LN),
and the cell cycle distribution by flow cytometry (not
The BLM antibody has been characterized (Ishov et al.,
1999; Neff et al., 1999; Zhong et al., 1999). As expected, it
failed to stain BS fibroblasts (Fig. 1, a and b), but identi-
fied 10–30 nuclear foci in WI-38 fibroblasts (Fig. 1, d–k),
confirming its specificity. To semiquantitatively assess
BLM foci, we scored the fraction of WI-38 cells with
discernible foci per nucleus (Fig. 1 c). Quiescent cells had
faint diffuse nuclear staining (not shown), but
10 faint foci (Fig. 1 c). This staining pattern persisted as
cells progressed through G1 (Q,8; Fig. 1 c). However, as
cells progressed through S (after release from HU), the
number (Fig. 1 c) and intensity (not shown) of BLM foci
rose, increasing until most cells were in late S or G2
(HU,8; Fig. 1 c). At this time, half the cells had
erally 20–40) bright BLM foci, which declined as cells en-
tered the next cell cycle (HU,10; Fig. 1 c). The intensity
and number of BLM foci were always heterogeneous, pos-
sibly due to the unavoidable loss of tight synchrony or the
dynamic nature of the foci. We did not detect BLM in nu-
cleoli, as reported for some cells (Yankiwski et al., 2000).
In sharp contrast to BLM foci, PML NBs, identified by a
PML antibody, did not vary in number (10–30 per nucleus)
or staining intensity, whether cells were in G0 or late S/G2
(Fig. 1, d–k). Similar results (invariant PML staining) were
obtained when G0 cells were compared with cells in G1 or
early S (not shown).
Although most (60–90%) PML NBs stained for BLM
during late S/G2, many were devoid of BLM at other cell
cycle stages. By contrast, most (80–90%) BLM foci stained
for PML, regardless of cell cycle position. The few BLM
foci that apparently lacked PML may indicate rare BLM
localization outside PML NBs, or failure of the antibody
to recognize PML in all NBs. Whatever the case, the ma-
jority of BLM colocalized with PML in human HCA2
The Journal of Cell Biology, Volume 153, 2001
normal fibroblasts, HT1080 fibrosarcoma, SAOS osteo-
sarcoma, and VA-13 SV40-transformed fibroblasts (not
shown). The exception was NB4 cells, which express a
dominant negative form of PML and show abnormal PML
organization into small aggregates or microspeckles (de
The et al., 1991; Mu et al., 1994). As reported by Zhong et
al. (1999), BLM showed mostly diffuse staining in NB4 nu-
clei (see Fig. 7 r), suggesting that PML is important, if not
essential, for BLM focus formation.
These results indicate that PML NBs are present
throughout the cell cycle, whereas BLM associates with
these structures as it is expressed, predominantly during S
is related to
, which functions in HR (Har-
mon and Kowalczykowski, 1998), a process that provides a
mechanism for repairing DNA during late S and G2
(Thompson and Schild, 1999). Thus, the BLM/PML foci
that form in late S/G2 might participate in HR to repair
spontaneous DNA damage that must be resolved before
mitosis. Therefore, we asked whether PML NBs also con-
tained hRAD51 or RP-A. These proteins interact, and are
critical for HR and HR repair (Baumann and West, 1997;
Golub et al., 1998; Kanaar et al., 1998; Thompson and
Schild, 1999). Moreover, BLM was recently shown to be
capable of interacting with RP-A (Brosh et al., 2000).
In quiescent cells, hRAD51 immunostaining was largely
diffuse throughout the nucleus, but
tained hRAD51 foci (Fig. 2 a), 70–80% of which localized
to PML NBs (Fig. 2, a–d). As cells approached late S/G2,
hRAD51 staining intensity increased, and
10 distinct RAD51/PML foci. More than half the
RAD51 foci also contained BLM (Fig. 2, e–h), and, in
about a third of the cells, 80–90% of the BLM foci con-
tained hRAD51. Western blotting showed that hRAD51
was detectable in quiescent cells, but expression increased
about fivefold as cells approached late S/G2 (not shown).
30% of nuclei con-
40% of nuclei
Figure 1. Cell cycle–dependent localization of
BLM. Cells were synchronized, immunostained
for BLM or PML, stained for nuclear DNA
(DAPI), and pulsed (1 h) with [3H]thymidine to
determine the percentage of cells in S phase (%
LN), as described in Materials and Methods. (a
and b) BLM antibody specificity. Proliferating
BS fibroblasts (HG2654, shown; GM11492F, not
shown) were stained with DAPI (a) to visualize
nuclei and the anti-BLM antibody (b). (c) BLM
foci during the cell cycle. WI-38 cells were ar-
rested in G0 (Q) and then stimulated with serum
for 8 h (Q,8) to enrich for cells in mid-G1. Alter-
natively, cells were arrested at the G1/S bound-
ary (HU) and released for varying intervals to
enrich for cells in mid-S (HU,4), late S/G2
(HU,8), G2/M/early G1 (HU,10), or G1/early S
(HU,12). The percent of LN was determined in
parallel cultures. Nuclei (?200 per data point)
were scored for the presence of ?10 BLM foci.
(d–k) BLM and PML were identified by im-
munostaining using fluorescein isothiocyanate
(green) or Texas red secondary antibodies. Red
and green fluorescent images were superim-
posed (MERGE). Nuclei were identified by
DAPI staining. (d) PML localization in quies-
cent cells. (e) BLM localization in quiescent
cells. (f) Merged image of PML and BLM local-
ization in quiescent cells. (g) DAPI staining of
nuclei in d–f. (h) PML localization in cells in late
S/G2. (i) BLM localization in cells in late S/G2.
(j) Merged image of PML and BLM localization
in cells in late S/G2. (k) DAPI staining of nuclei
in h–k. Bars, ?10 ?m.
Bischof et al.
BLM and DNA Damage
Thus, as cells progressed through late S and G2, an in-
creasing fraction assembled nuclear foci that contained
PML, BLM, and hRAD51. These results are summarized
in Table I. Because 40% of cells in late S/G2 had RAD51
50% of which contained BLM, and 80–90% of
BLM foci in late S/G2 localized to PML NBs, we deduce
that roughly 15–20% of late S/G2 nuclei contained all
three proteins (PML, RAD51, RAD51).
RP-A was evident as diffuse nuclear staining in quies-
cent cells, showing no obvious localization with PML or
BLM (not shown). However, during late S/G2 10–20% of
nuclei showed RP-A staining in a fraction of PML NBs
(Table I). In those nuclei with focal RP-A staining, 20–
30% of BLM-positive foci also stained positive for RP-A
(Fig. 2, i–l). Thus, as cells progressed through late S/G2,
10–20% had nuclei in which 20–30% of the foci contained
BLM and RP-A.
The results indicate that BLM localizes to PML NBs with
hRAD51, and to a lesser extent RP-A, during late S/G2.
BLM Associates with the Nuclear Matrix and hRAD51
PML resides in the nuclear matrix (Stuurman et al., 1992),
which focally concentrates nuclear processes such as RNA
transcription and splicing, and DNA replication and repair
(Spector, 1993; Lamond and Earnshaw, 1998). It is not
known whether all components of the PML NB are bound
to the nuclear matrix, or whether some components are
only loosely held to the matrix by PML.
To determine how BLM associates with the PML NB,
we prepared nuclear matrices from proliferating WI-38
cells using either of two methods: (a) standard high-salt
extraction of nuclei, or (b) extraction at lower ionic
strength followed by amine modification, which avoids the
potential artifact of nonspecific salt precipitation (Wan et
al., 1999). Proteins in the matrix and other fractions were
analyzed by Western blotting. Regardless of the prepara-
tion method, BLM was found predominantly in the nu-
clear matrix fraction, cofractionating with the nuclear ma-
trix marker protein lamin B (Spector, 1993; Lamond and
Earnshaw, 1998; Wan et al., 1999; Fig. 3, a and b). BLM
also fractionated with the nuclear matrix in VA-13 and
HT1080 tumor cells (not shown).
BLM also interacted with hRAD51 in vitro (Fig. 3, c and
d), suggesting that the colocalization of BLM and hRAD51
was a direct protein–protein interaction. Purified recombi-
nant GST (control) or GST-BLM (Fig. 3 c) bound to glu-
Figure 2. BLM, PML, hRAD51, and RP-A
localization in cells in G0 or late S/G2. WI-38
cells were made quiescent (Quiescent), or
released from a G1/S arrest for 8 h (Late
S/G2). Cells were stained for BLM, PML,
hRAD51, or RP-A, nuclei were visualized
(DAPI), and fluorescent images were super-
imposed (MERGE) as described in the
legend to Fig. 1. (a–d) hRAD51 and PML
localization in quiescent cells. (e–h)
hRAD51 and BLM localization in cells in
late S/G2. (i–l) RP-A and BLM localiza-
tion in cells in late S/G2. Bars, ?10 ?m.
Table I. Nuclear Foci Formed in Synchronized Human Fibroblasts
Percentage of nuclei with ?10 foci containing:
Cell cycle position PMLBLM RAD51RP-A PML?BLMPML?RAD51PML?BLM?RAD51*
WI-38 cells were synchronized, and the PML, BLM, hRAD51, and RP-A proteins were detected by immunofluorescence as described in Materials and Methods.
*The fraction of nuclei containing PML, BLM, and RAD51 was deduced from the degree of PML-BLM, PML-hRAD51, and BLM-hRAD51 colocalization as described in the text.
The Journal of Cell Biology, Volume 153, 2001
tathione-Sepharose and incubated with nuclear lysates from
SAOS-2 cells, which express high levels of hRAD51. The nu-
clear lysates were pretreated with DNase and ethidium bro-
mide to degrade DNA and disrupt protein–DNA interac-
tions. Proteins retained on the Sepharose were eluted with
glutathione and analyzed by Western blotting. GST-BLM,
but not GST, retained 30–40% of the hRAD51 in the lysate
(Fig. 3 d). By contrast, neither PARP nor Ku70, nuclear
DNA repair proteins that bind DNA, were retained by GST
or GST-BLM (Fig. 3 d). The lack of association of PARP or
Ku70, and resistance of the interaction between hRAD51
and GST-BLM to DNase or ethidium bromide, suggest that
the interaction is not mediated by DNA. BLM and hRAD51
could also be coimmunoprecipitated from nuclear lysates by
an anti-RAD51 antibody (Fig. 3 e). Despite this apparent in-
teraction between BLM and hRAD51 in cell nuclei, we could
not reliably coimmunoprecipitate BLM and hRAD51 using
an anti-BLM antibody. This failure may be due to disruption
of the BLM–hRAD51 complex by the anti-BLM antibody.
Nonetheless, our immunolocalization and biochemical data
suggest that BLM interacts with hRAD51, although whether
this is the case in cells is not yet conclusive.
Together, the immunofluorescence and biochemical re-
sults suggest that BLM may be a component of a dynamic
nuclear matrix–based complex that resides in the PML NB
and may participate in HR DNA repair during G2.
BLM Increases in Response to DNA Damage
To explore the idea that BLM plays a role in DNA repair,
we exposed proliferating cells to IR (5 Gy x-ray). IR
causes single and double strand DNA breaks, engaging
both the G1 and G2 checkpoints in normal human cells,
resulting in G1 and G2 transient cell cycle arrests or delays
(Kaufmann and Kies, 1998).
BLM mRNA was quantified using real-time RT-PCR
(Heid et al., 1996) and QM as a constitutively expressed con-
trol mRNA (Dimri et al., 1996). IR induced a modest, tran-
sient rise in BLM mRNA, amounting to a fourfold increase
within 2 h, before returning to the unirradiated (control)
level (Fig. 4 a). BLM protein also increased 2–4 h after IR,
Figure 3. BLM associates with the nuclear ma-
trix and hRAD51. Nuclear matrices were pre-
pared from proliferating WI-38 cells by high salt
extraction (NaCl), or low salt extraction and
amine modification (NH2SO4). After extraction,
30 ?g of protein was analyzed from whole cell
(Total), nuclear (Nuclear), and cytoplasmic (Cy-
tosol) lysates, and the supernatants (S) and nu-
clear matrix pellets (P). Proteins were analyzed
for BLM, ?-tubulin (Tubulin; cytosolic marker),
lamin B (nuclear matrix marker), PARP, and
Ku70 (DNA-associated) by Western blotting.
(a–b) Results of two independent fractionations.
(c) Recombinant GST-BLM. GST-BLM was
produced by baculovirus in insect cells. Nuclear
proteins from infected cells were bound to glu-
tathione-Sepharose, the resin was transferred to
a column, and bound proteins (200 ng) were
eluted and analyzed by silver-stained SDS-
PAGE. (d) hRAD51 associates with BLM. GST
or GST-BLM, bound to glutathione-Sepharose
beads, were incubated with SAOS-2 nuclear ly-
sates (Input) and transferred to a column. After
washing, proteins were eluted from the columns
(GST, Eluted; GST-BLM, Eluted), and proteins
resistant to elution were released by boiling in
SDS-PAGE sample buffer (GST, Boiled; GST-
BLM, Boiled). Input, eluted, and released pro-
teins were analyzed for BLM, hRAD51, PARP,
and Ku70 by Western blotting. (e) hRAD51
coimmunoprecipitates with BLM from nuclear ly-
sates. Nuclear lysates from SOAS-2 cells (Input)
were precleared and immunoprecipitated with
nonspecific (IgG) or anti-hRAD51 (?-RAD51)
antibody, and the immunoprecipitates were ana-
lyzed for BLM and hRAD51 by SDS-PAGE and
Western blotting, as described in Materials and
Bischof et al. BLM and DNA Damage
but in contrast to the mRNA, continued to accumulate for
8–10 h, peaking at 10-fold over the control level. Peak BLM
levels persisted for 4–6 h (12–14 h after IR; Fig. 4 b) before
declining to the control level (24 h after IR; not shown). A
lower dose (1 Gy) of IR induced less BLM (threefold over
control), but a higher dose (10 Gy) did not increase BLM fur-
ther (Fig. 4 c). Peak BLM induction by IR coincided with the
arrest of cell proliferation, detected by flow cytometry. Unir-
radiated cultures maintained a cell cycle distribution typical
of asynchronous populations (55% G1, 36% S, 9% G2/M;
Fig. 4 d). Irradiated cultures, by contrast, accumulated cells in
G1 (53%) and G2/M (42%) within 12 h, at which time fewer
than 5% of cells were in S phase (Fig. 4 d). Because the cells
have a finite replicative life span, even the early passage cul-
tures used here contain 15–20% senescent cells, which have a
G1 DNA content (Campisi, 1997). Thus, the fraction of irra-
diated cells that transiently arrested in G1 was likely ?40%,
whereas the fraction that arrested in G2 was likely ?50%.
Cells resumed proliferation 24 h after IR (not shown). These
results raise the possibility that BLM is induced by DNA
damage. Alternatively, because BLM is expressed predomi-
nantly in late S/G2, its accumulation after IR may reflect the
accumulation of cells in G2.
BLM Response to DNA Damage Depends on
a G2 Delay
To distinguish between these possibilities, we X-irradiated
(5 Gy) quiescent cells, then stimulated them with serum.
Under these conditions, cells remain in G1 for 24 h, with-
out transiently arresting in G2 (Kaufmann and Kies, 1998;
not shown). BLM levels did not change (Fig. 5 a). Similar
results were obtained with irradiated senescent cells,
which do not enter, much less arrest in, G2 (not shown).
We also irradiated proliferating cells and immediately
gave them caffeine, which abolishes the G2 delay (Busse
et al., 1977; Tolmach et al., 1977; Schlegel and Pardee,
1986). Caffeine-treated cells showed a small (two- to
threefold) transient rise in BLM, but no sustained BLM
accumulation (Fig. 5 b) and little or no G2 delay. 3 h after
IR, irradiated cultures had few, if any, mitotic figures, indi-
cating failure to leave G2. By contrast, caffeine-treated ir-
radiated cultures had half the mitotic index of unirradiated
controls 3 h after IR, and two to three times the mitotic in-
dex of controls 6–12 h after IR (Fig. 5 b), indicating that
many cells entered mitosis with little or no G2. Finally, we
treated cells with other DNA damaging agents, only some
of which cause a G2 delay. Bleomycin and etoposide cause
DNA DSBs and G1 and G2 delays (Kaufmann and Kies,
1998). Bleomycin increased BLM six- to eightfold, very
similar to the effects of IR (Fig. 5 c). Etoposide also in-
creased BLM six- to eightfold, albeit with slower kinetics
(Fig. 5 d), perhaps reflecting its slower action. In contrast,
BLM was unchanged by UVC (1.6 J/m2/s; Fig. 5 e) or hy-
drogen peroxide (550 ?M; not shown), which cause pre-
dominantly base damage and single strand breaks and ar-
rest normal cells primarily in G1 (Kaufmann and Kies,
1998). Thus, the rise in BLM after IR most likely reflected
the transient G2 arrest that occurs when proliferating cells
BLM Response to DNA Damage Is Independent of AT
Mutated and p53
Further evidence that the G2 delay is responsible for the
rise in BLM caused by IR came from cells deficient in AT
mutated (ATM), the gene defective in AT. AT is a heredi-
tary cancer-prone syndrome characterized by loss of the
G1, but not the G2, DNA damage checkpoint. DNA DSBs
cause AT cells to accumulate in G2 for an extended inter-
val (Beamish et al., 1994). BLM protein was two- to four-
fold less abundant in proliferating AT fibroblasts (strain
AT-2SF; Tobias et al., 1984) compared with wild-type cells
(WI-38), consistent with our observation that BLM is
lower in slow growing cell strains compared with more
rapidly dividing strains. Despite the low basal level, IR (5
Figure 4. BLM responds to IR. Proliferating WI-
38 cells were X-irradiated (IR) with 5 Gy (a, b,
and d) or 0–10 Gy (c). RNA and protein were iso-
lated, or cells were harvested for flow cytometry,
at the indicated intervals (h) thereafter. BLM
mRNA was measured by quantitative PCR using
QM as a control; BLM protein was assessed by
Western blotting using ?-tubulin (Tubulin) as a
control. A value of one was assigned to the nor-
malized levels of BLM mRNA and protein in
unirradiated cells (0 h). Autoradiograms of the
Western analyses are shown above the histograms.
(a) BLM mRNA after IR. (b) BLM protein after
IR. (c) IR dose response. Cells were analyzed for
BLM protein 4 h (autoradiogram) or 8 h (auto-
radiogram, histogram) after irradiation. (d) Cell
cycle arrest after IR. Cells were analyzed for
DNA content by flow cytometry. The G1 (2N)
and G2 (4N) peaks are indicated and the fraction
of cells in G1, S, and G2/M is given in the text.
The Journal of Cell Biology, Volume 153, 2001
Gy x-ray) increased BLM ?10-fold in AT cells (Fig. 5 f),
similar to the magnitude of increase in wild-type cells (Fig.
4 b). However, in contrast to wild-type cells, BLM re-
mained elevated for ?24 h (compared with 12–14 h for
wild-type cells), consistent with their longer G2 delay.
The accumulation of BLM after IR was also indepen-
dent of p53. WI-38 cells were rendered p53-deficient by
expressing the E6 viral oncogene, which accelerates p53
degradation (Scheffner et al., 1990). IR (5 Gy x-ray)
caused BLM accumulation (Fig. 5 f), and a transient G2
arrest (not shown), in proliferating E6-expressing cells,
very similar to its effects on control cells.
BLM Foci Increase after DNA Damage
Several proteins that participate in DSB repair form nu-
clear foci in response to IR. These foci, known as IRIF
(Maser et al., 1997), are present in undamaged cells, but
increase in number after IR. One type of IRIF contains
hRAD51 and RP-A, and is thought to carry out HR repair
of DSBs (Haaf et al., 1995; Golub et al., 1998). Because
BLM formed foci and localized with hRAD51 and RP-A
in a fraction of undamaged nuclei, we asked whether BLM
foci, like RAD51/RP-A IRIF, increased after IR.
BLM foci were heterogeneously distributed in asynchro-
nous cultures, with few nuclei containing ?10 foci (Fig. 6 a).
However, after X-irradiation (5 Gy) nuclei with ?10 BLM
foci rose, whereas those with <10 foci declined, in a time-
(Fig. 6 a) and dose- (not shown) dependent manner. 10 h af-
ter IR, nuclei with ?10 BLM foci were four- to fivefold
more prevalent than in control cultures. Moreover, at this
time 15–20% of irradiated nuclei had ?20 BLM foci,
whereas such nuclei were rare in controls (Fig. 6 b). The IR-
induced peak in BLM foci (Fig. 6 a) coincided with the IR-
induced peak in BLM protein and G2 delay (Fig. 4). Etopo-
side similarly increased BLM foci coincident with BLM
protein and a G2 delay (not shown). By contrast, BLM foci
did not rise after UV irradiation (Fig. 6 b), which did not in-
crease BLM expression (Fig. 5 e) and delays cells in G1
(Kaufmann and Kies, 1998). Compared with controls, UV-
irradiated cultures had more nuclei with ?10 BLM foci, and
fewer with 11–20 BLM foci (Fig. 6 b), consistent with their
more prominent G1 delay. Thus, BLM foci increased in re-
sponse to agents that cause DSBs, similar to IRIF, and the
increase coincided with the G2 delay.
IR increased the number of BLM foci in proliferating
SV40-transformed cells (VA-13; not shown), which lack
p53 function, MO59J cells (not shown), which lack the cat-
alytic subunit of DNA-dependent protein kinase (DNA-
PK), and AT-2SF cells (Fig. 6 c), which lack ATM. AT cul-
tures accumulated two- to threefold more nuclei with ?20
BLM foci than wild-type cultures 10 h after IR (Fig. 6 c),
consistent with their prolonged peak of BLM expression
Figure 5. BLM response to DNA damage depends on the G2 delay. WI-38 (a–e), AT-2SF, or WI38-E6 (f) cells were treated with the
indicated agents while quiescent (a) or proliferating (b–f). Protein lysates were prepared from untreated cells (0 h) or at the indi-
cated times after treatment (h), and analyzed for BLM and ?-tubulin (Tubulin; control) by Western blotting. (a) Quiescent cells
were X-irradiated (5 Gy) and immediately stimulated with serum. Parallel cultures were pulsed for 24 h with [3H]thymidine to determine
the percentage of cells that synthesized DNA (% LN). (b) Proliferating cells were X-irradiated (5 Gy) and immediately given me-
dium containing 5 mM caffeine. Parallel cultures were analyzed for mitotic figures (Mitotic index; 1,000 nuclei/point). The mitotic
index of the unirradiated culture was given a value of 1. (c) Cells were given bleomycin (10 ?g/ml) for 1 h in serum-containing me-
dium. (d) Cells were given etoposide (10 ?M) for 1 h in serum-containing medium. (e) Cells were irradiated with UVC (1.6 J/m2/s)
in PBS and returned to serum-containing medium. (f) Proliferating AT-2SF or WI38-E6 cells were X-irradiated (5 Gy), protein lysates
were prepared, and BLM protein level was normalized to ?-tubulin.
Bischof et al. BLM and DNA Damage
(Fig. 5 f) and G2 delay (Beamish et al., 1994). Interest-
ingly, AT cells also accumulate more RAD51 IRIF than
wild-type cells (Maser et al., 1997).
BLM and PML Are Components of IRIF
The rise in BLM foci caused by IR and prevalence of BLM
(Fig. 6 c) and hRAD51 (Maser et al., 1997) foci in irradiated
AT cells suggest that BLM might be a component of
RAD51 IRIF. To test this idea, we immunostained cells for
BLM, hRAD51, and RP-A 10–12 h after IR, when the G2
delay, BLM expression, and BLM foci were maximal. At
this time, ?50% of nuclei had ?20 hRAD51 and/or RP-A
foci. Greater than 90% of the RAD51 foci costained for
BLM and ?90% of BLM foci costained for hRAD51 (Fig.
7, a–d). Thus, there was near complete colocalization of
BLM and RAD51 to the same foci after IR. RP-A foci were
more numerous than BLM/RAD51 foci (?30 per nucleus;
Fig. 7 f), with ?60% containing BLM; ?70% of BLM foci
contained RP-A (Fig. 7, e–h). BLM remained localized to
PML NBs after IR, evident by the high coincidence of BLM
and PML staining (Fig. 7, i–l). These results indicate that
BLM and PML are components of RAD51 IRIF and a frac-
tion of RP-A IRIF. Because RP-A foci outnumbered BLM/
RAD51 foci after IR, RP-A may function in some IR re-
sponses that are distinct from those in which BLM and
hRAD51 participate, or act with different kinetics.
BLM was not essential for RAD51 IRIF formation be-
cause BS cells formed abundant RAD51 foci in response
to IR (Fig. 7, m–o). BS cells formed 2–2.5-fold more
RAD51 foci as normal cells, consistent with reduced or de-
layed repair. By contrast, few IRIF formed in NB4 cells,
which express a dominant negative form of PML. In con-
trol NB4 cells, hRAD51 and BLM were largely dispersed
throughout the nucleus (Fig. 7, p and r). IR induced a few
hRAD51 and BLM foci, but most of the hRAD51 and
BLM remained disperse (Fig. 7, q and s). This result sug-
gests that, in contrast to BLM, PML is important for IRIF
BLM Associates with Single-stranded DNA
RAD 51/RP-A IRIF are important for HR DNA repair
and, after IR, accumulate at sites of single-stranded DNA
(Raderschall et al., 1999) or unscheduled DNA synthesis
(Haaf et al., 1999). If BLM and PML are components of
RAD51 IRIF and participate in repair, they should accu-
mulate at sites of repair. BrdU, a thymidine analogue, has
been used to visualize sites of single-stranded DNA and
presumptive DSB repair (Raderschall et al., 1999). BrdU
is inaccessible to an anti-BrdU antibody when present in
duplex DNA, but readily accessible to the antibody when
the DNA is denatured or single stranded. We used BrdU
to determine whether BLM/PML foci associate with sin-
gle-stranded DNA after IR-induced damage.
Cells were grown for two doublings in the presence of
BrdU. Proliferating BrdU-labeled cells, fixed and stained
under nondenaturing conditions, showed no significant
staining (Fig. 8 a), confirming that the antibody does not
detect BrdU in duplex DNA. The same cells showed the
expected heterogeneous pattern of BLM foci (Fig. 8 b), in-
dicating that BrdU did not perturb BLM localization.
When BrdU-labeled cells were X-irradiated (5 Gy), BrdU
foci appeared (Fig. 8 c), peaking 8–10 h after IR. At this
time, ?50% of the cells had multiple BrdU foci. These foci
were not due to apoptosis because there was no evidence
of PARP degradation, which precedes apoptotic DNA
fragmentation, for at least 10 h after IR (Fig. 8 d). More-
over, BrdU foci appeared to be a specific response to
DSBs because UV induced few if any BrdU foci (not
shown). Thus, in agreement with Raderschall et al. (1999),
BrdU foci formed primarily in response to DNA DSBs,
where they presumably identify sites of repair.
To determine whether BLM localized with BrdU, we
costained for BLM and BrdU 10–12 h after IR. A signifi-
cant fraction of BLM localized to BrdU foci. In 30–50% of
cells with BrdU foci, 80–90% of the BrdU foci costained
for BLM and 80–90% of the BLM foci in these cells co-
stained for BrdU (Fig. 8, e–g). In the remaining cells with
BrdU foci, BLM was present in a variable fraction of
BrdU foci, ranging from 30 to 50% (Fig. 8, h–m). More-
over, PML also associated with a significant fraction of
BrdU foci (Fig. 8, n–p). Thus, in response to IR, BLM and
PML NBs, like RAD51 and RP-A (Raderschall et al.,
Figure 6. BLM focus formation after DNA damage. Proliferating
WI-38 (a–c) and AT-2SF (c) cells were X-irradiated (5 Gy) or UV-
irradiated (1.6 J/m2/s) and immunostained for BLM at the indi-
cated intervals thereafter. BLM foci were counted in 200 nuclei
per point. (a) BLM foci increase after IR. Nuclei were scored for
the presence of ?10 or ?10 BLM foci. (b) Effect of IR versus
UV. Cells were unirradiated (?IR) or irradiated with X-rays
(?IR) or UV (?UV). 10 h later, nuclei were scored for the pres-
ence of 0–10, 11–20, or ?20 BLM foci. (c) BLM foci formation in
irradiated AT cells. Proliferating AT-2SF or WI-38 cells were
X-irradiated. 10 h later, nuclei were scored for the presence of
11–20 or ?20 BLM foci.
The Journal of Cell Biology, Volume 153, 2001
1999), assembled at sites of single-stranded DNA, which
presumably are undergoing HR repair.
Defects in BLM have severe physiological consequences
in humans, the most prominent of which is premature
death due to cancer. At the cellular level, defects in BLM
cause genomic instability, particularly chromosome aber-
rations and SCEs. The phenotypes associated with BLM
deficiency, and the homology to RECQ, suggest that BLM
may function in an HR DNA repair pathway that resolves
spontaneous and induced DNA damage. In support of this
idea, we confirmed that BLM is expressed primarily in late
S/G2, when HR repair is operational, and found it was in-
duced by agents that cause DNA DSBs and engage the G2
checkpoint. We also found that BLM is a component of
RAD51 IRIF, which are thought to be important for the
repair of DSBs by HR. Our data suggest that BLM and
RAD51 interact, and BLM was found to interact with RP-A
(Brosh et al., 2000). BLM foci contained RAD51, and to
a lesser extent RP-A, and formed in undamaged cells dur-
ing G2. BLM foci containing RAD51 also formed in IR-
damaged cells, where they localized to sites of presump-
tive repair. During the cell cycle and after IR, the majority
of BLM associated with PML NBs, a matrix-based orga-
nizing center for many nuclear processes. PML appeared
to be important for organizing BLM and hRAD51 into
foci, particularly IRIF. Together, our results suggest that
BLM participates in normal G2 functions and the G2
Figure 7. BLM localizes with hRAD51, RP-A,
and PML after IR. Proliferating WI-38, BS
HG2654, and NB4 cells were X-irradiated (5 Gy)
where indicated. 10 h after irradiation, the cells
were immunostained for BLM, PML, hRAD51,
or RP-A; nuclei were visualized (DAPI); and flu-
orescent images were superimposed (MERGE)
as described in the legend to Fig. 1. (a–d) BLM
and hRAD51 localization in irradiated WI-38
cells. (e–h) BLM and RP-A localization in irra-
diated WI-38 cells. (i–l) BLM and PML localiza-
tion in irradiated WI-38 cells. (m–o) hRAD51
localization in irradiated BS cells. (p–s) hRAD51
and BLM localization in unirradiated (?IR) and
irradiated (?IR) NB4 cells. Bars, ?10 ?m.
Bischof et al. BLM and DNA Damage
DNA damage response, and implicates the PML NB in as-
sembling BLM, hRAD51, and probably other proteins
that are important for both processes.
Function in Undamaged Cells
BLM foci formed predominantly in late S and G2 and in-
creased during the G2 delay induced by DNA DSB, sug-
gesting that BLM participates in a G2 function. In undam-
aged cells, BLM may be needed for proper termination of
DNA replication. As the genome is duplicated, excessive
recombination must be suppressed and sister chromatids
must be disentangled to ensure accurate mitosis. Consis-
tent with this idea, BS cells accumulate replication inter-
mediates (Ockey and Saffhill, 1986; Lonn et al., 1990).
BLM may also regulate mitotic recombination during G2.
SCEs are mediated by hRAD51 and hRAD54 and can re-
pair DNA lesions by HR at the end of S phase (Sonoda et
al., 1999). Whether BLM stimulates or suppresses recom-
Figure 8. BLM and PML localize to sites of putative
DNA repair after IR. Proliferating WI-38 were
labeled for two doublings with BrdU and X-irra-
diated (5 Gy) where indicated. 10–12 h after irradia-
tion, the cells were fixed under nondenaturing
conditions and immunostained for BrdU, BLM,
and PML. Nuclei were visualized (DAPI)
and fluorescent images were superimposed
(MERGE) as described in the legend to Fig. 1.
(a) BrdU staining in unirradiated cells. (b) BLM
foci in unirradiated, BrdU-labeled cells. (c) BrdU
and DAPI staining in irradiated cells. The BrdU
and DAPI images were merged. (d) PARP integ-
rity after irradiation. BrdU-labeled cells were left
untreated (?IR), X-irradiated (?IR), or treated
with Fas ligand (Fas; positive control for apopto-
sis). 10 h later, protein lysates were prepared and
analyzed for PARP by Western blotting. (e–m)
BLM and BrdU localization in irradiated cells.
e–g show a cell with 80–90% colocalization of
BLM and BrdU foci; 30–50% of cells with BrdU
foci showed this staining pattern. h–m show cells
in which BLM and BrdU colocalized to varying
degrees. (n–p) PML and BrdU localization in ir-
radiated cells. Bar, ?10 ?m.
The Journal of Cell Biology, Volume 153, 2001
bination is not yet known. BLM may suppress recombina-
tion during normal cell cycle progression, but promote re-
combination during repair of DSBs. BLM also localizes
with RP-A (Walpita et al., 1999) and RAD51 (Moens et
al., 2000) in mammalian spermatocytes undergoing mei-
otic prophase (Walpita et al., 1999). This localization and
the male infertility characteristic of BS suggest that BLM
may also function during meiosis.
Response to DNA Damage and Role in HR
BS cells have been reported to be more sensitive than wild-
type cells to radiation, particularly in late S and G2 (Aurias
et al., 1985; Hall et al., 1986). BLM protein increased and
assembled into foci specifically in response to agents that
caused DNA DSBs. This induction and assembly was not a
primary response to DSBs. Rather, it reflected engage-
ment of the G2 checkpoint and the arrest of cells in G2.
DNA DSBs are potentially catastrophic lesions because
they can lead to unequal distribution of DNA to daughter
cells. Mammalian cells are thought to repair DNA DSBs
primarily by nonhomologous end joining (Karanjawala et
al., 1999), but recent evidence shows that HR is also im-
portant for resolving such damage (Kanaar et al., 1998;
Thompson and Schild, 1999). Repair by HR requires ex-
tensive regions of sequence homology, which are provided
by the undamaged sister chromatid. Hence, HR repair oc-
curs almost exclusively in S or G2. HR repair is carried out
by the RAD52 complex, which includes hRAD51 and RP-A
(Kanaar et al., 1998; Thompson and Schild, 1999). Several
lines of evidence suggest that BLM participates in HR re-
pair: (a) BLM is most highly expressed in late S and G2,
when HR occurs; (b) BLM interacted with hRAD51,
which is critical for HR; (c) BLM colocalized with
hRAD51, and to a lesser extent RP-A (also important for
HR), in both undamaged and damaged cells; (d) many of
the RAD51 foci that formed after IR contained BLM; and
(e) BLM foci localized to sites of presumptive repair (sin-
gle-stranded DNA) after IR.
BLM IRIF were more prevalent in AT cells, relative to
wild-type cells. AT cells are known to accumulate fewer
RAD50/MRE11 IRIF and more RAD51 IRIF than wild-
type cells (Maser et al., 1997). The RAD50–MRE11 com-
plex, with proteins like DNA-PK, participates in DSB
repair by nonhomologous end joining. ATM, the gene
defective in AT, encodes a kinase that phosphorylates p53
in response to IR, increasing its half-life and affinity for
target genes (Banin et al., 1998; Canman et al., 1998). Be-
cause p53 activation is impaired, AT cells do not undergo
a G1 checkpoint arrest, but rather enter a prolonged G2
delay after IR (Beamish et al., 1994). This delay coincided
with a prolonged period of elevated BLM and BLM IRIF.
Because RAD50/MRE11 foci are deficient in damaged
AT cells and BLM/RAD51 foci persist, DSBs normally re-
paired by nonhomologous end joining may be repaired by
HR in AT cells. Our finding that BLM IRIF form inde-
pendent of ATM, p53, and DNA-PK are consistent with
the idea that BLM IRIF participate in HR repair.
Our findings do not rule out additional roles for BLM, for
example in other DNA repair pathways. The recent finding
that BLM is a component of a supercomplex containing the
RAD50–MRE11 complex and proteins important for tran-
scription-coupled repair (Wang et al., 2000) suggests that
BLM may participate in multiple DNA transactions.
Role of PML NBs
PML may play an important role in assembling BLM/
hRAD51 foci during normal G2 progression and during
the G2 delay induced by IR. Regardless of cell cycle posi-
tion or DNA damage, the majority of BLM localized to
PML NBs. In undamaged cells, some BLM/PML NBs also
contained hRAD51. However, after irradiation the major-
ity of BLM/PML foci contained hRAD51, and a substan-
tial fraction of these foci also contained RP-A. Thus, IRIF
contained BLM and PML, in addition to RAD51 and RP-A.
This was not the case in NB4 cells, in which PML is dys-
functional. In undamaged NB4 cells, neither BLM nor
hRAD51 was organized into foci. Rather, both proteins
were dispersed throughout the nucleus. After NB4 cells
were irradiated, neither BLM nor RAD51 IRIF foci
formed. Thus, PML appears to be essential for the forma-
tion of IRIF. By contrast, BLM does not appear to be es-
sential for PML NB formation (Ishov et al., 1999; Zhong et
al., 1999). Likewise, BLM did not appear to be required
for RAD51 IRIF formation, since BS cells formed RAD51
foci in response to IR.
PML NBs appear to be sites of BLM, RAD51, and RP-A
assembly during late S/G2, and after irradiation. Both
BLM and PML localized to sites of single-stranded DNA
and presumptive repair after damage by IR. Thus, one
function of PML NBs may be to assemble RAD51/BLM
IRIF to carry out HR DNA repair during S/G2 in undam-
aged cells and during G2 in damaged cells. It is also possi-
ble that PML NBs are involved in assembling RAD50/
MRE11 IRIF, but this remains to be determined.
In the absence of PML function, the NB structure is dis-
rupted and cells either arrest growth or undergo apoptosis
(de The et al., 1991; Mu et al., 1994; Le et al., 1996; Wang
et al., 1998a,b). One possibility is that these cellular re-
sponses are due to a failure to resolve DNA damage at the
end of S phase or during G2. The association of BLM and
PML with the nuclear matrix and with IRIF and sites of
presumptive DSB repair suggest that PML NBs assemble
a matrix-based complex containing BLM and hRAD51
which functions as a HR recombinosome to repair sponta-
neous and induced DNA DSBs.
We thank Drs. E. Blakely, J. German, O. Pereira-Smith, and D. Chen for
cell lines, and D. Chen for antibodies and helpful discussions.
Supported by grants from the National Institute on Aging (AG11658)
to J. Campisi under Department of Energy contract DE-AC0376SF00098
to the University of California, and by the Sloan-Kettering Institute and
May Samual Rudin Family Foundation to N.A. Ellis.
Submitted: 1 May 2000
Revised: 6 February 2001
Accepted: 20 February 2001
Aurias, A., J.L. Antoine, R. Assathiany, M. Odievre, and B. Dutrillaux. 1985.
Radiation sensitivity of Bloom’s syndrome lymphocytes during S and G2
phases. Cancer Genet. Cytogenet. 16:131–136.
Banin, S., L. Moyal, S. Shieh, Y. Taya, C.W. Anderson, L. Chessa, N.I. Smoro-
dinsky, C. Prives, Y. Reiss, Y. Shiloh, and Y. Ziv. 1998. Enhanced phosphory-
lation of p53 by ATM in response to DNA damage. Science. 281:1674–1677.
Baumann, P., and S.C. West. 1997. The human Rad51 protein: polarity of
strand transfer and stimulation by hRP-A. EMBO (Eur. Mol. Biol. Organ.)
Bischof et al. BLM and DNA Damage
Beamish, H., K.K. Khanna, and M.F. Lavin. 1994. Ionizing radiation and cell
cycle progression in ataxia telangiectasia. Radiat. Res. 138:130–133.
Brosh, R.M., J.L. Li, M.K. Kenny, J.K. Karow, M.P. Cooper, R.P. Kureekattil,
I.D. Hickson, and V.A. Bohr. 2000. Replication protein A physically inter-
acts with the Bloom’s syndrome protein and stimulates its helicase activity.
J. Biol. Chem. 275:23500–23508.
Busse, P.M., S.K. Bose, R.W. Jones, and L.J. Tolmach. 1977. The action of caf-
feine on X-irradiated HeLa cells. II. Synergistic lethality. Radiat. Res. 71:
Campisi, J. 1997. The biology of replicative senescence. Eur. J. Cancer. 33:703–
Canman, C.E., D.S. Lim, K.A. Cimprich, Y. Taya, K. Tamai, K. Sakaguchi, E.
Appella, M.B. Kastan, and J.D. Sciliano. 1998. Activation of the ATM ki-
nase by ionizing radiation and phosphorylation of p53. Science. 281:1677–
Compton, D.A., T. Yen, and D.W. Cleveland. 1991. Identification of a novel
centromere/kinetochore-associated protein using monoclonal antibodies
generated against human mitotic chromosome scaffolds. J. Cell Biol. 112:
Davey, S., C.S. Han, S.A. Ramer, J.C. Klassen, A. Jacobson, A. Eisenberger,
K.M. Hopkins, H.B. Lieberman, and G.A. Freyer. 1998. Fission yeast rad121
regulates cell cycle checkpoint control and is homologous to the Bloom’s
syndrome disease gene. Mol. Cell. Biol. 18:2721–2728.
de The, H., C. Lavau, A. Marchio, C. Chomienne, L. Degos, and A. Dejean.
1991. The PML-RAR alpha fusion mRNA generated by the t(15;17) translo-
cation in acute promyelocytic leukemia encodes a functionally altered RAR.
Dignam, J.D., R.M. Lebovitz, and R.G. Roeder. 1983. Accurate transcription
initiation by RNA polymerase II in a soluble extract from isolated mamma-
lian nuclei. Nucleic Acids Res. 11:1475–1489.
Dimri, G.P., E.E. Hara, and J. Campisi. 1994. Regulation of two E2F-related
genes in presenescent and senescent human fibroblasts. J. Biol. Chem. 269:
Dimri, G.P., X. Lee, G. Basile, M. Acosta, G. Scott, C. Roskelley, E.E. Me-
drano, M. Linskens, I. Rubelj, O.M. Pereira-Smith, M. Peacocke, and J.
Campisi. 1995. A novel biomarker identifies senescent human cells in cul-
ture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA. 92:9363–9367.
Dimri, G.P., A. Testori, M. Acosta, and J. Campisi. 1996. Replicative se-
nescence, aging and growth regulatory transcription factors. Biol. Signals.
Dimri, G.P., K. Itahana, M. Acosta, and J. Campisi. 2000. Regulation of a se-
nescence checkpoint response by the E2F1 transcription factor and p14/
ARF tumor suppressor. Mol. Cell. Biol. 20:273–285.
Dutertre, S., M. Ababou, R. Onclercq, J. Delic, B. Chatton, C. Jaulin, and M.
Amor-Gueret. 2000. Cell cycle regulation of the endogenous wild type
Bloom’s syndrome DNA helicase. Oncogene. 19:2731–2738.
Ellis, N.A., and J. German. 1996. Molecular genetics of Bloom’s syndrome.
Hum. Mol. Genet. 5:1457–1463.
Ellis, N.A., J. Groden, T.Z. Ye, J. Straughen, D.J. Lennon, S. Ciocci, M.
Proytcheva, and J. German. 1995. The Bloom’s syndrome gene product is
homologous to RecQ helicases. Cell. 83:655–666.
Fukuchi, K., G.M. Martin, and R.J. Monnat. 1989. Mutator phenotype of
Werner syndrome is characterized by extensive deletions. Proc. Natl. Acad.
Sci. USA. 86:5893–5897.
German, J. 1993. Bloom syndrome: a Mendelian prototype of somatic muta-
tional disease. Medicine. 72:393–406.
Gharibyan, V., and H. Youssoufian. 1999. Localization of the Bloom syndrome
helicase to punctate nuclear structures and the nuclear matrix and regulation
during the cell cycle: comparison with the Werner’s syndrome helicase. Mol.
Golub, E.I., R.C. Gupta, T. Haaf, M.S. Wold, and C.M. Radding. 1998. Interac-
tion of human hRAD51 recombination protein with single-stranded DNA
binding protein, RPA. Nucleic Acids Res. 26:5388–5393.
Goto, M. 1997. Hierarchical deterioration of body systems in Werner’s syn-
drome: implications for normal ageing. Mech. Ageing Dev. 98:239–254.
Gray, M.D., J.C. Shen, A.S. Kamath-Loeb, A. Blank, B.L. Sopher, G.M. Mar-
tin, J. Oshima, and L.A. Loeb. 1997. The Werner syndrome protein is a
DNA helicase. Nat. Genet. 17:100–103.
Gray, M.D., L. Wang, H. Youssoufian, G.M. Martin, and J. Oshima. 1998.
Werner helicase is localized to transcriptionally active nucleoli of cycling
cells. Exp. Cell Res. 242:487–494.
Haaf, T., E.I. Golub, G. Reddy, C.M. Radding, and D.C. Ward. 1995. Nuclear
foci of mammalian HRAD51 recombination protein in somatic cells after
DNA damage and its localization in synaptonemal complexes. Proc. Natl.
Acad. Sci. USA. 92:2298–2302.
Haaf, T., E. Raderschall, G. Reddy, D.C. Ward, C.M. Radding, and E.I. Golub.
1999. Sequestration of mammalian Rad51-recombination protein into mi-
cronuclei. J. Cell Biol. 144:11–20.
Halbert, C.L., G.W. Demes, and D.A. Galloway. 1992. The E7 gene of human
papillomavirus type 16 is sufficient for immortalization of human epithelial
cells. J. Virol. 66:2125–2134.
Hall, E.J., M.J. Marchese, M.B. Astor, and T. Morse. 1986. Response of cells of
human origin, normal and malignant, to acute and low dose rate irradiation.
Int. J. Radiat. Oncol. Biol. Phys. 12:655–659.
Hanada, K., T. Ukita, Y. Kohno, K. Saito, J. Kato, and H. Ikeda. 1997. RecQ
DNA helicase is a suppressor of illegitimate recombination in Escherichia
coli. Proc. Natl. Acad. Sci. USA. 94:3860–3865.
Harmon, F.G., and S.C. Kowalczykowski. 1998. RecQ helicase, in concert with
RecA and SSB proteins, initiates and disrupts DNA recombination. Genes
Heid, C.A., J. Stevens, K.J. Livak, and P.M. Williams. 1996. Real time quantita-
tive PCR. Genome Res. 6:986–994.
Ishov, A.M., A.G. Sotnikov, D. Negorev, O.V. Vladimirova, N. Neff, T. Kami-
tani, E.T.H. Yeh, J.F. Strauss, and G.G. Maul. 1999. PML is critical for
ND10 formation and recruits the PML-interacting protein Daxx to this nu-
clear structure when modified by SUMO-1. J. Cell Biol. 147:221–233.
Kanaar, R., J.H. Hoeijmakers, and D.C. van Gent. 1998. Molecular mecha-
nisms of DNA double strand break repair. Trends Cell Biol. 12:483–489.
Karanjawala, Z.E., U. Grawunder, C.L. Hsieh, and M. Lieber. 1999. The non-
homologous DNA end joining pathway is important for chromosome stabil-
ity in primary fibroblasts. Curr. Biol. 9:1501–1504.
Karow, J.K., R.K. Chakraverty, and I.D. Hickson. 1997. The Bloom’s syndrome
gene product is a 3?-5? DNA helicase. J. Biol. Chem. 272:30611–30614.
Kaufmann, W.K., and P.E. Kies. 1998. DNA signals for G2 checkpoint re-
sponse in diploid human fibroblast. Mutat. Res. 400:153–167.
Kitao, S., I. Ohsugi, K. Ichikawa, M. Goto, Y. Furuichi, and A. Shimamoto.
1998. Cloning of two new human helicase genes of the RecQ family: biologi-
cal significance of multiple species in higher eukaryotes. Genomics. 54:443–
Lamond, A.I., and W.C. Earnshaw. 1998. Structure and function in the nucleus.
Le, X.F., P. Yang, and K.S. Chang. 1996. Analysis of the growth and transfor-
mation suppressor domains of promyelocytic leukemia gene, PML. J. Biol.
Lonn, U., S. Lonn, U. Nylen, G. Winblad, and J. German. 1990. An abnormal
profile of DNA replication intermediates in Bloom’s syndrome. Cancer Res.
Lu, K.H., R.A. Levine, and J. Campisi. 1989. c-ras-Ha gene expression is regu-
lated by insulin or insulinlike growth factor and by epidermal growth factor
in murine fibroblasts. Mol. Cell. Biol. 9:3411–3417.
Martin, G.M., J. Oshima, M.D. Gray, and M. Poot. 1999. What geriatricians
should know about the Werner syndrome. J. Am. Geriatr. Soc. 47:1136–
Maser, R.S., K.J. Monsen, B.E. Nelms, and J.H. Petrini. 1997. hMre11 and
hRad50 nuclear foci are induced during the normal cellular response to
DNA double-strand breaks. Mol. Cell. Biol. 17:6087–6096.
Miozzo, M., P. Castorina, P. Riva, L. Dalpra, A.M. Fuhrman Conti, L. Volpi,
T.S. Hoe, A. Khoo, J. Wiegant, C. Rosenberg, and L. Larizza. 1998. Chro-
mosomal instability in fibroblasts and tumors from 2 sibs with Rothmund-
Thomson syndrome. Int. J. Cancer. 77:504–510.
Moens, P.B., R. Freire, M. Tarsounas, B. Spyropoulos, and S.P. Jackson. 2000.
Expression and nuclear localization of BLM, a chromosome stability protein
mutated in Bloom’s syndrome, suggest a role in recombination during mei-
otic prophase. J. Cell Sci. 113:663–672.
Mu, Z.M., K.V. Chin, J.H. Liu, G. Lozano, and K.S. Chang. 1994. PML, a
growth suppressor disrupted in acute promyelocytic leukemia. Mol. Cell.
Neff, N.F., N.A. Ellis, T.Z. Ye, J. Noonan, K. Huang, M. Sanz, and M.
Proytcheva. 1999. The DNA helicase activity of BLM is necessary for the
correction of the genomic instability of Bloom syndrome cells. Mol. Biol.
Ockey, C.H., and R. Saffhill. 1986. Delayed DNA maturation, a possible cause
of the elevated sister-chromatid exchange in Bloom’s syndrome. Carcino-
Pucillo, C., S. Salzano, S. Pepe, M. Vitale, S. Formisano, and G. Rossi. 1990.
Regulation of the expression of the low-affinity IgE receptor (Fc epsilon
RII) in the human monocyte-like cell line U-937 by phorbol esters and IgE.
Int. Arch. Allergy Appl. Immunol. 93:330–337.
Puranam, K.L., and P.J. Blackshear. 1994. Cloning and characterization of
RECQL, a potential human homologue of the Escherichia coli DNA heli-
case RecQ. J. Biol. Chem. 269:29838–29845.
Raderschall, E., E.I. Golub, and T. Haaf. 1999. Nuclear foci of mammalian re-
combination proteins are located at single-stranded DNA regions formed af-
ter DNA damage. Proc. Natl. Acad. Sci. USA. 96:1921–1926.
Scheffner, M., B.A. Werness, J.M. Huibregtse, A.J. Levine, and P.M. Howley.
1990. The E6 oncoprotein encoded by human papillomavirus types 16 and
18 promotes the degradation of p53. Cell. 63:1129–1136.
Schlegel, R., and A.B. Pardee. 1986. Caffeine-induced uncoupling of mitosis
from the completion of DNA replication in mammalian cells. Science. 232:
Seki, M., H. Miyazawa, S. Tada, J. Yanagisawa, T. Yamaoka, S. Hoshino, K.
Ozawa, T. Eki, M. Nogami, K. Okumura, et al. 1994. Molecular cloning of
cDNA encoding human DNA helicase Q1 which has homology to Esche-
richia coli Rec Q helicase and localization of the gene at chromosome 12p12.
Nucleic Acids Res. 22:4566–4573.
Sonoda, E., M.O. Sasaki, C. Morrison, Y. Yamaguchi-Iwal, M. Takata, and S.
Takeda. 1999. Sister-chromatid exchanges are mediated by homologous re-
combination in vertebrate cells. Mol. Cell. Biol. 19:5166–5169.
Spector, D.L. 1993. Macromolecular domains in the cell nucleus. Annu. Rev.
Cell Biol. 9:265–315.
Starr, D.G., J.P. McClure, and J.M. Connor. 1985. Non-dermatological compli-
The Journal of Cell Biology, Volume 153, 2001 Download full-text
cations and genetic aspects of the Rothmund-Thomson syndrome. Clin.
Stewart, E., C.R. Chapman, F. Al-Khodairy, A.M. Carr, and T. Enoch. 1997.
Rqh11, a fission yeast gene related to the Bloom’s and Werner’s syndrome
genes, is required for reversible S phase arrest. EMBO (Eur. Mol. Biol. Or-
gan.) J. 16:2682–2692.
Stuurman, N., A. de Graaf, A. Floore, A. Josso, B. Humbel, L. de Jong, and R.
van Driel. 1992. A monoclonal antibody recognizing nuclear matrix-associ-
ated nuclear bodies. J. Cell Sci. 101:773–784.
Tada, S., J. Yanagisawa, T. Sonoyama, A. Miyajima, M. Seki, M. Ui, and T.
Enomoto. 1996. Characterization of the properties of a human homologue
of Escherichia coli RecQ from xeroderma pigmentosum group C and from
HeLa cells. Cell Struct. Funct. 21:123–132.
Thompson, L.H., and D. Schild. 1999. The contribution of homologous recom-
bination in preserving genome integrity in mammalian cells. Biochimie. 81:
Tobias, C.A., E.A. Blakely, P.Y. Chang, L. Lommel, and R. Roots. 1984. Re-
sponse of sensitive human ataxia and resistant T-1 cell lines to accelerated
heavy ions. Br. J. Cancer. 6:175–185.
Tolmach, L.J., R.W. Jones, and P.M. Busse. 1977. The action of caffeine on
X-irradiated HeLa cells. I. Delayed inhibition of DNA synthesis. Radiat.
Vennos, E.M., and W.D. James. 1995. Rothmund-Thomson syndrome. Derma-
tol. Clin. 13:143–150.
Walpita, D., A.W. Plug, N.F. Neff, J. German, and T. Ashley. 1999. Bloom’s
syndrome protein, BLM, colocalizes with replication protein A in meiotic
prophase nuclei of mammalian spermatocytes. Proc. Natl. Acad. Sci. USA.
Wan, K.M., J.A. Nickerson, G. Krockmalnic, and S. Penman. 1999. The nuclear
matrix prepared by amine modification. Proc. Natl. Acad. Sci. USA. 96:933–
Wang, Y., D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, and J. Qin. 2000. BASC, a
super complex of BRCA1-associated proteins involved in the recognition
and repair of aberrant DNA structures. Genes Dev. 14:927–939.
Wang, Z., D. Ruggero, S. Ronchetti, M. Zhong, M. Gaboli, R. Rivi, and P. Pan-
dolfi. 1998a. PML is essential for multiple apoptotic pathways. Nat. Genet.
Wang, Z.G., L. Delva, M. Gaboli, R. Rivi, M. Giorgio, C. Cordon-Cardo, F.
Grosveld, and P. Pandolfi. 1998b. Role of PML in cell growth and the retin-
oic acid pathway. Science. 279:1547–1551.
Watt, P.M., and I.D. Hickson. 1996. Failure to unwind causes cancer. Genome
stability. Curr. Biol. 6:265–267.
Watt, P.M., I.D. Hickson, R.H. Borts, and E.J. Louis. 1997. SGS1, a homologue
of the Bloom’s and Werner’s syndrome genes, is required for maintenance
of genome stability in Saccharomyces cerevisiae. Genetics. 144:935–945.
Wessel, G.M., and D.R. McClay. 1986. Two embryonic, tissue-specific mole-
cules identified by a double-label immunofluorescence technique for mono-
clonal antibodies. J. Histochem. Cytochem. 34:703–706.
Yankiwski, V., R.A. Marciniak, L. Guarente, and N.F. Neff. 2000. Nuclear
structure in normal and Bloom syndrome cells. Proc. Natl. Acad. Sci. USA.
Yu, C.E., J. Oshima, Y.H. Fu, E.M. Wijsman, F. Hisama, R. Alisch, S. Mat-
thews, J. Nakura, T. Miki, S. Ouais, et al. 1996. Positional cloning of the
Werner’s syndrome gene. Science. 272:258–262.
Zhong, S., P. Hu, T.Z. Ye, R. Stan, N.A. Ellis, and P.P. Pandolfi. 1999. A role
for PML and the nuclear body in genomic stability. Oncogene. 18:7841–7847.