From the Imperial Cancer Research Fund Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom
Received for publication, October 17, 2000, and in revised form, February 8, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bloom's syndrome (BS) is an autosomal recessive
disorder that predisposes individuals to a wide range of cancers. The
gene mutated in BS, BLM, encodes a member of the RecQ
family of DNA helicases. The precise role played by these enzymes in
the cell remains to be determined. However, genome-wide
hyper-recombination is a feature of many RecQ helicase-deficient cells.
In eukaryotes, a central step in homologous recombination is catalyzed
by the RAD51 protein. In response to agents that induce DNA
double-strand breaks, RAD51 accumulates in nuclear foci that are
thought to correspond to sites of recombinational repair. Here, we
report that purified BLM and human RAD51 interact in vitro
and in vivo, and that residues in the N- and C-terminal
domains of BLM can independently mediate this interaction. Consistent
with these observations, BLM localizes to a subset of RAD51 nuclear
foci in normal human cells. Moreover, the number of BLM foci and the extent to which BLM and RAD51 foci co-localize increase in response to
ionizing radiation. Nevertheless, the formation of RAD51 foci does not
require functional BLM. Indeed, in untreated BS cells, an abnormally
high proportion of the cells contain RAD51 nuclear foci. Exogenous
expression of BLM markedly reduces the fraction of cells containing
RAD51 foci. The interaction between BLM and RAD51 appears to have been
evolutionarily conserved since the C-terminal domain of Sgs1, the
Saccharomyces cerevisiae homologue of BLM, interacts with
yeast Rad51. Furthermore, genetic analysis reveals that the
SGS1 and RAD51 genes are epistatic indicating that they operate in a common pathway. Potential roles for BLM in the
RAD51 recombinational repair pathway are discussed.
Germline mutations in the BLM gene give rise to
Bloom's syndrome (BS),1 a
rare disorder associated with stunted growth, facial sun sensitivity, immunodeficiency, fertility defects, and a greatly elevated increase in
the occurrence of a wide range of cancers (1). BLM encodes a
159-kDa protein that is a member of the RecQ family of DNA helicases (2). This highly conserved family of proteins is required for the
maintenance of genomic stability in all organisms (3). In humans, five
RecQ helicases have been identified. In addition to BLM,
mutations in two other genes encoding RecQ helicases in humans have
been associated with disease conditions: WRN and
RECQ4, being defective in Werner's syndrome and
Rothmund-Thomson syndrome, respectively (4, 5). Werner's syndrome is
primarily associated with premature aging, and Rothmund-Thomson
syndrome with skin and skeletal abnormalities, but both disorders also
give rise to an elevated incidence of cancers (6, 7). All RecQ family members contain a catalytic helicase domain that comprises seven highly
conserved motifs found in many DNA and RNA helicases (3). Outside of
this helicase domain, the RecQ family proteins show little sequence
conservation. In BLM, these non-conserved domains are located both N-
and C-terminal to the helicase domain and comprise ~650 and 450 amino
acids, respectively. It is likely that these non-conserved
domains are important in functionally differentiating the roles of the
different RecQ helicases within the cell by either providing additional
enzymatic functions, such as the exonuclease activity dependent upon
the N-terminal domain of WRN (8, 9), or by mediating interactions with
other proteins (10).
Cells from BS patients display genomic instability, the diagnostic
feature being an increase in the frequency of sister chromatid exchanges (SCEs) (11). Sonoda et al. (12) recently
demonstrated that SCE formation requires homologous recombination (HR).
Moreover, chicken BLM The general mechanism for repair of DNA double-strand breaks (DSBs) by
HR has been conserved in evolution. The central step involves the
pairing of the DSB with homologous sequences to facilitate the exchange
of DNA strands. In bacteria, this process is mediated by RecA, which
forms a nucleoprotein filament on single-stranded DNA formed as a
result of exonucleolytic processing of the DSB to generate
single-stranded DNA tails. This nucleoprotein filament facilitates the
search for homologous sequences and provides a structure within which
DNA strand exchange occurs. In eukaryotes, essentially the same
reaction is performed by RAD51, which is structurally related to RecA
(21-24). In Saccharomyces cerevisiae, the RAD51
gene is a member of the RAD52 epistasis group, and
rad51 mutants display defects in mitotic and meiotic
recombination and sensitivity to ionizing radiation, highlighting the
key role that RAD51 plays in both HR and recombinational repair of DNA
strand breaks (25). Studies on RAD51 in higher eukaryotes have been hampered by the fact that mice with a targeted disruption of the RAD51 gene die early during embryogenesis (26, 27). However, studies with early mouse embryos suggest that loss of RAD51
renders cells sensitive to ionizing radiation (26). Additionally,
RAD51 mutant chicken cells, maintained by expression of
human RAD51 (hRAD51) under the control of a regulable promoter,
accumulate chromosomal breaks following repression of hRAD51 synthesis
(28). This suggests a role for hRAD51 in the repair of DNA breaks in undamaged cycling cells, most likely those arising during DNA replication. Treatment of mammalian cells with agents that induce DSBs,
such as ionizing radiation, induces localization of Rad51 to nuclear
foci that are believed to correspond to multiprotein complexes engaged
in recombinational repair (29). However, the precise composition of
these recombinational repair centers is unknown.
In this study, we have examined the possibility that the Bloom's
syndrome gene product functions in the RAD51 recombinational repair
pathway given the defects in recombination displayed by BS cells. BLM
and hRAD51 were found to directly interact in vitro and
co-immunoprecipitate from nuclear extracts. Consistent with these data,
BLM forms nuclear foci, a subset of which, co-localize with hRAD51.
Furthermore, the degree to which these two proteins co-localize to
nuclear foci increases in response to ionizing radiation. The
interaction between BLM and hRAD51 appears to have been evolutionarily
conserved since Sgs1 physically associates with yeast RAD51, and
genetic analysis reveals that the genes encoding these two proteins are epistatic.
Cell Lines--
The SV40-transformed normal human fibroblast
cell line, WI-38 (obtained from ATCC), was used as a representative
normal human cell line. The GM08505 cell line is an SV40-transformed
fibroblast cell line from a BS patient (obtained from NIGMS, National
Institutes of Health, Bethesda, MD) and contains a BLM
homozygous frameshift mutation at residue 739 resulting in premature
truncation of the protein (2). PSNF5 cells were derived from a clone of
GM08505 cells stably transfected with pcDNA3/BLM. Western blotting
using an anti-BLM antiserum, IHIC33 (10), was used to confirm that BLM
was stably expressed by this line. Functional complementation of the BS
phenotype was assessed by SCE frequency analysis which showed that
PSNF5 cells have a near-normal SCE frequency. Derivation and
characterization of these cells will be described
elsewhere.2 All three cell
lines were routinely cultured in Bacterial and Yeast Strains--
The Escherichia coli
BL21(DE3) strain was obtained from New England Biolabs. Gene
disruptions were done in the S. cerevisiae YP-1 strain
(his4-R leu2 MATa-URA3-MATa ura3-52 ade2-101 lys2). The
SGS1 open reading frame was replaced with LYS2 as
described previously (15). The RAD51 open reading frame was
replaced with LEU2. The two-hybrid screen was performed in
EGY48 (MATa his3 trp1 ura3-52
lex(leu2)3a).
Plasmids--
The generation of pcDNA3/BLM and pYES/BLM-NC
has been described previously (10). The plasmid pGEX-4T1 (Amersham
Pharmacia Biotech) was used for the expression of glutathione
S-transferase (GST) fusion peptides. Portions of the
BLM cDNA that encode various N- and C-terminal fragments
of BLM were amplified by PCR. Sense and antisense primers contained the
seven terminal 5' sense and 3' antisense codons, respectively, of each
desired fragment. The antisense primer had an additional in-frame
antisense stop codon. EcoRI and XhoI sites were
also engineered into the sense and antisense primers, respectively, to
allow in-frame cloning of the PCR fragments into pGEX-4T-1. For the
two-hybrid screen, the LexA-fusion DNA binding domain vector (pEG202),
the Antibodies--
Polyclonal (IHIC33) and monoclonal (BFL103)
anti-BLM antibodies have been described previously (10). Anti-Rad51
antiserum was a kind gift from Dr. Steve West (Imperial Cancer Research Fund, London, United Kingdom). Anti-bromodeoxyuridine (BrdUrd) antibodies were from Dako.
Expression and Purification of Recombinant
Proteins--
Recombinant BLM and BLM-NC proteins were expressed in
yeast from the expression plasmids pYES/BLM and pYES/BLM-NC (10), respectively, and purified using nickel chelate affinity chromatography as previously described (31). Purified recombinant hRAD51 was a kind
gift from Dr. Steve West. GST fusion peptides were expressed in and
purified from BL21 (DE3) cells as previously described (10).
Nuclear Extracts--
Nuclear extracts were prepared as
previously described (10) from exponentially growing WI-38 cells or
cells that had been arrested in 5 µg/ml aphidicolin for 14 h.
Yeast Two-hybrid Screen--
The HeLa cDNA activation
library in pJG45 was transformed into EGY48, which already contained
pEG202/BLM (residues 966-1417) and pSH1834 using a modified method of
Gietz et al. (32). Transformants were selected for uracil,
histidine, and tryptophan prototrophy on 2% glucose, before being
replica-plated to 2% galactose, 1% raffinose plates containing 40 µg/ml 5-bromo-4-chloro-3-indolyl Indirect Immunofluorescence Analysis--
Intracellular
localization of BLM and hRAD51 was visualized using the methodologies
described previously (10) using BFL103 and anti-Rad51 antibodies,
respectively, in exponentially growing cells or cells that had been
irradiated with 10 Gy or cultured with 5 µg/ml aphidicolin for
14 h. Quantitation of nuclear foci was determined from 100-200
cells for each treatment. For the detection of sites of DNA synthesis
and hRAD51, cells grown on coverslips were incubated for 10 min at
37 °C in 150 µM BrdUrd, fixed in 4% paraformaldehyde
in 250 mM HEPES (pH 7.4) at 4 °C for 20 min,
permeabilized with 0.1% Triton X-100 at 4 °C for 20 min, followed
by three washes with phosphate-buffered saline. Cells were blocked in
phosphate-buffered saline containing 10% fetal calf serum for 15 min
and then incubated for 1 h with anti-RAD51 antibodies and for 30 min with fluorescein isothiocyanate-conjugated secondary antibody
(Dako). Incorporated BrdUrd was detected by a second fixation step of
20 min at 4 °C in 4% paraformaldehyde in 250 mM HEPES
(pH 7.4) followed by a 15-min incubation in 8% paraformaldehyde in 250 mM HEPES (pH 7.4). Cells were then incubated in 4 M HCl for 15 min at 37 °C followed by five washes in
phosphate-buffered saline. Incorporated BrdUrd was immunolabeled using
mouse monoclonal antibodies against BrdUrd and anti-mouse IgG
conjugated with Cy3 (Sigma). DNA was visualized by Hoechst staining.
Immunoprecipitations and Western Blotting--
Typically,
nuclear extracts prepared from 108 cells were used for each
immunoprecipitation. Immunoprecipitations were carried out as described
previously (10) with the exception that anti-Rad51 immunoprecipitates
were captured. IHIC33 anti-serum was used to detect the presence of BLM
using conventional Western blotting techniques.
Far Western Analysis--
This technique was performed as
described previously (10) using recombinant full-length hRAD51 and
either full-length recombinant BLM, BLM-NC, or GST fusion peptides
containing N- or C-terminal fragments of BLM. Bound hRAD51 was detected
using anti-RAD51 antiserum.
Measurement of Growth of S. cerevisiae--
Serial dilutions of
exponentially growing yeast cultures were spotted onto either YPD
plates or YPD plates containing drug and incubated for 2-3 days at
30 °C. Alternatively, exponentially growing yeast cultures were
diluted into pre-warmed YPD medium or YPD medium containing drug, and
the cell number monitored, using a Coulter counter (Sysmex F-820), over
a period of 6 h. Growth inhibition was determined by calculating
the percentage difference in cell number between the culture in
drug-containing medium versus the control culture in
drug-free YPD. Experiments were performed a minimum of three times on
two independent segregants of each strain.
BLM and hRAD51 Directly Interact via the N- and C-terminal Domains
of BLM--
The helicase domain in BLM, in common with the human WRN
and RECQ4 proteins, is flanked by relatively large N- and C-terminal domains (3) that are likely important in functionally distinguishing these larger members of the RecQ helicase family. To analyze the role
of these domains in BLM, we performed a yeast two-hybrid screen of a
HeLa cDNA library using the C-terminal domain (residues 966-1417)
of BLM as bait. One positive clone, isolated independently three times,
was found to encode hRAD51. The interaction appeared specific since
hRAD51 did not interact with two nonspecific control bait proteins
(Fig. 1A). Further mapping
revealed that the C-terminal 150 amino acids of BLM (residues
1267-1417) were sufficient to mediate an interaction with hRAD51.
Since only a fragment of BLM had been used in the two-hybrid screen,
and interactions using this system could in principle be mediated by
adaptor proteins, we used Far Western analysis with purified
recombinant BLM and hRAD51 to determine if the full-length proteins
were able to interact directly with each other. Far Western analysis
revealed that the full-length proteins could interact (Figs.
1B and 2B) and that the interaction was specific
since hRAD51 did not interact with either of two control proteins used
in this assay, GST and MBP (Fig. 1C and data not
shown). Using affinity-purified GST fusion peptides containing various
portions of the C-terminal domain of BLM, the final 100 residues,
representing amino acids 1317-1417 of BLM, were identified as being
sufficient to mediate an interaction with hRAD51 (Fig. 1C),
thus providing independent confirmation of the location of the hRAD51
interaction domain on BLM identified by the yeast two-hybrid system
(Fig. 1A).
To investigate the possibility of additional hRAD51 interaction domains
on BLM, Far Western analysis was performed between hRAD51 and a GST
fusion protein containing the first 212 amino acids of BLM. hRAD51 was
also able to specifically interact with this protein (Fig.
2A), indicating that BLM
contains at least two domains with which hRAD51 can independently
associate with (Figs. 1C and 2A). In contrast to
full-length BLM, a purified recombinant mutant protein, BLM-NC, which
consists of residues 213-1266 of BLM and therefore lacks both the N-
and C-terminal hRAD51 interaction domains on BLM, was unable to bind
hRAD51. This suggests that no additional portions of BLM interact with hRAD51 (Fig. 2B).
BLM and hRAD51 Form a Complex in Vivo and Co-localize to Nuclear
Foci in Response to DNA Damage--
In response to DNA DSBs generated
by ionizing radiation, hRAD51 localizes to nuclear foci that are
thought to correspond to sites of ongoing repair (Table
I, part A) (29). BLM also forms nuclear
foci in undamaged normal cells (Table I, part A) that have been shown
previously to correspond to PML nuclear bodies (33-35). In an
asynchronous population of WI-38 cells, a human fibroblast cell line
derived from a normal individual, the proportion of cells containing
BLM foci and the average number of BLM foci/cell was found to increase
in response to ionizing radiation (Table I, part A) (Fig.
3). We therefore examined the possibility
that BLM might co-localize with hRAD51 foci in response to ionizing radiation. In a population of unirradiated WI-38 cells, 16% of the
cells contained a mean of 2.1 co-localizing BLM and hRAD51 foci/nucleus
(Fig. 3 and Table I). Three hours after 10 Gy of
The demonstration that BLM and hRAD51 directly interact in
vitro and co-localize to nuclear foci suggested that these two proteins might exist as part of a multiprotein complex in
vivo. To investigate this further, we analyzed whether BLM and
hRAD51 could be co-immunoprecipitated from WI-38 nuclear extracts. In extracts prepared from unirradiated or A High Proportion of Undamaged BS Cells Contain Sites of
Recombinational Repair--
The precise mechanism by which hRAD51
forms nuclear foci is unknown. We therefore determined if BLM was
required for hRAD51 to localize to nuclear foci. In the absence of
exogenous damage, 84% of GM08505 cells, a fibroblast cell line from a
BS patient, contained hRAD51 nuclear foci (Fig.
5A). In contrast, in an
asynchronous population of PSNF5 cells, a clone derived from GM08505
cells that expresses the BLM cDNA, only 30% of the
cells contained hRAD51 nuclear foci (Fig. 5A). A similar
proportion of hRAD51 nuclear foci-containing cells was also observed in
exponentially growing WI-38 cells (Table I, part A). In undamaged
normal cells, RAD51 foci have been shown to form predominantly in S
phase (37). Therefore, one possible explanation for the difference in
the proportion of GM08505 and PSNF5 cells containing hRAD51 foci might be an altered cell cycle distribution. However, fluorescence-activated cell sorting analysis of propidium iodide-stained GM08505 and PSNF5
cells revealed that the two cell lines had comparable proportions of
cells in G1, S, and G2 phases of the cell cycle
(Fig. 5B). Furthermore, the high proportion of GM08505 cells
containing hRAD51 foci suggests that hRAD51 foci can form outside S
phase. To confirm this, we pulse-labeled GM08505 and PSNF5 cells with
BrdUrd. Indirect immunofluorescence with anti-BrdUrd and anti-hRAD51
antibodies revealed that hRAD51 foci were clearly evident in non-S
phase cells in both GM08505 and PSNF5 cells (Fig. 5C).
Together, these data indicate that hRAD51 foci can form in the absence
of BLM and suggest that, in BS cells, DNA lesions (possibly DSBs) are formed that activate the hRAD51-dependent stress response
resulting in a constitutively high level of hRAD51 foci.
The S. cerevisiae RecQ Homologue, Sgs1, Also Interacts with
Rad51--
Since both RecQ helicases and Rad51 have been conserved
through evolution, and given that RecQ helicase mutants in yeast and humans give rise to defects in genetic recombination, we addressed the
question of whether Sgs1, the S. cerevisiae homologue of
BLM, interacts with yeast Rad51 in the two-hybrid system. Sgs1, like BLM, also contains N- and C-terminal domains that flank the core helicase domain (3). Although the C-terminal domains of Sgs1 and BLM
share little sequence homology, the C-terminal domain (residues
978-1447) of Sgs1 was found to specifically interact with yeast Rad51
(Fig. 6A). A region within
this domain containing residues 1299-1447 was sufficient to mediate an
interaction with yeast RAD51 (Fig. 6A), whereas a fragment
containing residues 1319-1447 of Sgs1 was not able to interact with
Rad51. This indicates that amino acid residues 1299-1318 of Sgs1 are
important for the interaction with Rad51.
We next investigated the possibility that the SGS1 and
RAD51 genes are epistatic. Mutants that lack either Sgs1 or
Rad51 are viable but are sensitive to agents that perturb DNA
replication or certain DNA damaging drugs. In particular,
sgs1 mutants are sensitive to the ribonucleotide reductase
inhibitor, hydroxyurea (HU), and the methylating agent, methyl
methanesulfonate (MMS) (Fig. 6, B and C), as has
been found by other workers (38, 39). rad51 mutants also
showed a similar magnitude of sensitivity to HU and MMS (Fig. 6,
B and C). However, deletion of both the
SGS1 and RAD51 genes did not appear to have any
additive or synergistic effect on either HU or MMS sensitivity,
suggesting that these two genes are epistatic. Together, these data
suggest that Sgs1 and Rad51 function as a complex in a common pathway.
The underlying cellular defects that give rise to BS remain
unclear. Cytogenetic analyses of BS cells suggest that either homologous recombination occurs at an increased frequency or that recombination events are aberrantly processed. In this study, we have
shown that BLM, the product of the gene defective in BS, forms a
complex with the recombination protein hRAD51. This provides the first
direct molecular link between BLM and the pathway for HR in human cells.
We have presented several lines of evidence that BLM and hRAD51
interact directly via residues in the N- and C-terminal domains of BLM,
consistent with the finding that BLM and hRAD51 form a complex in
nuclear extracts. Despite the lack of sequence homology between BLM and
Sgs1, outside of the helicase domain, the C-terminal domain of Sgs1 was
found to interact with yeast Rad51, indicating that the organization of
functional domains within these two RecQ helicases has been conserved
to some extent. The evolutionary conservation of an interaction between
RecQ helicases and RAD51 implies that these two classes of proteins
together perform a fundamentally important role during DNA metabolism.
The finding that SGS1 and RAD51 are epistatic is
in agreement with the findings of Gangloff et al. (40), who
demonstrated that deletion of RAD51 can rescue the synthetic
lethality of an sgs1 The stress response pathway leading to nuclear hRAD51 focus formation
is apparently constitutively activated in BS cells, but can be
suppressed by ectopic expression of BLM. These data suggest both that
BLM is not essential to induce the stress response pathway, and that a
basal level of unrepaired lesions persist in the absence of BLM. This
is consistent with the fact that BS cells are not hypersensitive to
ionizing radiation (41, 42) and indicate that the pathways for
repairing the majority of DSBs are essentially intact in BS cells.
Similarly, haploid sgs1 Not all hRAD51 foci contained detectable levels of BLM, suggesting that
BLM only acts upon a subset of DNA lesions processed by hRAD51. One
possibility is that BLM may process DSBs that occur at replication
forks since BLM has been shown to co-localize to replication foci (43).
Such a role would be consistent with the expression profile of BLM
through the cell cycle and the replication defects seen in BS cells.
DNA strand breaks could arise during replication when, for example, the
replication fork encounters a single-stranded DNA nick in the template
DNA (Fig. 7). The formation of a DSB
would result in collapse of the replication fork (Fig. 7). Indeed, it
has been postulated that such events occur every cell cycle in the
absence of exogenous DNA damage (44-46). The exact mechanism by which
replication is re-initiated following formation of a DSB remains to be
elucidated. However, it is becoming apparent that replication restart
is inextricably linked to recombination (44-46). In bacteria,
RecA-mediated strand invasion of the broken arm of the replication fork
into the intact chromosome to form a D-loop is thought to provide a
structure necessary for the re-initiation of replication by a pathway
involving the PriA helicase (46). In eukaryotes, RAD51 may serve a
similar purpose in allowing replication to reinitiate following
replication fork collapse (Fig. 7). The resulting Holliday junction
would then need to be removed. We suggest that hRAD51 recruits BLM to
perform this function, which could be achieved by either branch
migrating the junction toward the end of the chromosome or facilitate
resolution of the junction through recruitment of Holliday junction
resolvases (Fig. 7).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
cells
display elevated SCE levels that are partially dependent on
RAD54 (13). BLM therefore seems to function in the
regulation of HR events during replication. Consistent with this
notion, mutations in SGS1 or
rqh1+, the budding and fission yeast
RecQ homologues, respectively, also give rise to excessive
recombination events (14-16). In rqh1 mutants, inhibition
of DNA replication, in particular, stimulates this excessive
recombination (16). Further evidence of a role for RecQ helicases in HR
comes from the finding that RecQ, Sgs1, BLM, and WRN can all disrupt
four-way junctions, a structural mimic for the Holliday junction
intermediate formed during HR (17-20). Moreover, both BLM and WRN can
promote branch migration of Holliday junctions (19, 20).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimal essential medium
supplemented with 10% fetal bovine serum. HeLa S3 cells were
maintained in RPMI 1640 supplemented with 10% fetal bovine serum.
-galactosidase reporter plasmid (pSH1834) and the plasmid pJG45,
which contained the HeLa cDNA activation library, were kind gifts
from Dr. R. Brent. pLexA-MAX (30) and pHM12 (kindly provided by Drs. R. Finley and R. Brent) are derived from pEG202 and contain the entire
open reading frame of human MAX and 295 residues of Drosophila
melanogaster Cdc2 kinase, respectively. Portions of the
BLM or SGS1 cDNA that encode various
C-terminal regions of each protein were amplified by PCR and cloned
into pEG202. Sense and antisense primers contained the seven terminal
5' sense and 3' antisense codons, respectively, of each desired
fragment. The antisense primer had an additional in-frame antisense
stop codon. EcoRI and XhoI sites were also engineered into the sense and antisense primers, respectively, to allow
in-frame cloning of the PCR fragments into pEG202. The entire S. cerevisiae RAD51 open reading frame was amplified by PCR from
yeast genomic DNA and cloned directionally into pJG45 via
EcoRI and XhoI sites that were engineered into
the sense and antisense PCR primers, respectively.
-D-galactopyranoside
(X-gal). Plasmids from
-galactosidase positive colonies were
isolated and sequenced.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (32K):
[in a new window]
Fig. 1.
BLM and hRAD51 interact directly.
A, interaction in the yeast two-hybrid system between
hRAD51 and various fragments of BLM as indicated. Also shown are three
negative controls: MAX and HM12, which do not interact with hRAD51, and
an empty vector control, demonstrating that the shortest BLM fragment
that interacts with hRAD51 does not alone activate expression of
-galactosidase from the reporter plasmid. B, Far Western
analysis of full-length purified recombinant BLM and hRAD51.
Nitrocellulose membranes, to which BLM was transferred following
SDS-PAGE, were incubated with hRAD51 (+) or buffer alone (
) as
indicated above and then probed for the presence of bound hRAD51 by
Western blotting using an anti-hRAD51 antibody. The position of BLM on
the membranes is indicated on the left. C, Far
Western analysis of full-length purified hRAD51 and various GST fusion
peptides containing portions of BLM as indicated. Left
panel is a Coomassie Blue-stained gel showing the purified
GST fusion peptides alongside GST alone as indicated on the
left. The full-length GST fusion peptides are somewhat
unstable as demonstrated by the presence of degradation products.
Middle and right panels are the same
GST peptides transferred to nitrocellulose membranes and then probed
with hRAD51 (+) or buffer alone (
) as indicated above and then
Western-blotted for the presence of bound hRAD51 using an anti-hRAD51
antibody.
View larger version (33K):
[in a new window]
Fig. 2.
A portion of the N-terminal domain of BLM
binds to hRAD51. A, Far Western analysis of full-length
purified hRAD51 and a GST fusion peptide containing residues 1-212 of
BLM (GST-BLM). Left panel is a Coomassie
Blue-stained gel showing the purified GST-BLM fusion peptide alongside
GST alone, as indicated on the left. Middle and
right panels are the same proteins as shown in
the left panel transferred to nitrocellulose
membranes and then probed with hRAD51 (+) or buffer alone ( ), as
indicated above. The membranes were then Western-blotted for the
presence of bound hRAD51 using an anti-hRAD51 antibody. B,
Full-length BLM but not a fragment of BLM representing residues
213-1266 binds to hRAD51. Far Western analysis of hRAD51 against
full-length purified recombinant BLM and BLM-NC. Nitrocellulose
membranes, to which BLM and BLM-NC were transferred following SDS-PAGE,
were incubated with hRAD51 (right panel) as
indicated above and then probed for the presence of bound hRAD51 by
Western blotting using an anti-hRAD51 antibody. Left
panel shows a Western blot of an identical membrane using
anti-BLM antibodies and indicates the relative amounts of each protein
used. The positions of BLM and BLM-NC on the membranes are indicated on
the left.
-irradiation, these
figures increased significantly such that 24% of the population
contained a mean of 4.7 co-localizing BLM and hRAD51 foci/nucleus (Fig.
3 and Table I, part B). By 6 h following irradiation, the
proportion of cells containing BLM and hRAD51 co-localizing nuclear
foci had declined to almost the level seen in the untreated population
(18%). However, the mean number of co-localizing nuclear foci (3.4)
was still significantly higher than that in the untreated control
population (Table I, part B).
Quantitation of the proportion of cells containing BLM and hRAD51 foci
in untreated, irradiated, or aphidicolin-treated WI-38 cells (A) and
quantitation of nuclear foci containing both BLM and hRAD51 in
untreated, irradiated, or aphidicolin-treated WI-38 cells (B)
View larger version (16K):
[in a new window]
Fig. 3.
BLM and hRAD51 co-localize to nuclear foci in
response to -irradiation. Indirect
immunofluorescence of BLM (red) and hRAD51
(green) in untreated and irradiated WI-38 cells as indicated
on the left. In the merged image, co-localization is seen as
yellow. The panel labeled DNA
indicates the position of the nucleus, as judged by Hoechst
staining.
-irradiated WI-38 cells, we
were unable to reproducibly co-immunoprecipitate BLM using anti-hRAD51
antibodies (data not shown). One possible explanation for this is that
only a maximum of 24% of these cells were observed to contain BLM and
hRAD51 co-localizing foci (Table I, part B). We therefore sought a
treatment that would enrich for the number of cells containing BLM and
hRAD51 co-localizing foci. BLM is cell-cycle regulated and is most
highly expressed in the S and G2/M phases of the cell cycle
and is absent from G1 cells (36). Synchronization of cells
in early S phase using aphidicolin resulted in an increase in the
number of cells containing BLM and hRAD51 foci (Table I, part
A) and, concomitantly, a nearly 3-fold increase (41%
versus 16%) in the proportion of cells containing BLM and hRAD51 co-localizing foci (Table I, part B). Using nuclear extracts prepared from these aphidicolin-treated cells, we were able to readily
immunoprecipitate BLM using anti-hRAD51 antibodies suggesting that BLM
and hRAD51 exist as a complex in vivo at least under certain
circumstances (Fig. 4).
View larger version (38K):
[in a new window]
Fig. 4.
BLM and hRAD51 exist as a complex in nuclear
extracts. Western blot for the detection of BLM in
immunoprecipitates of nuclear extracts prepared either from untreated
or aphidicolin-arrested WI-38 cells. The immunoprecipitating antibody
used was either anti-hRAD51 or pre-immune sera as indicated above. The
position of BLM is indicated on the right.
View larger version (20K):
[in a new window]
Fig. 5.
The RAD51 stress response is constitutively
activated in BS cells. A, indirect immunofluorescence
of hRAD51 (pale blue) in GM08505 (upper
panel) and PSNF5 (lower panel) cells.
Shown also is the position of the nucleus of each cell (dark
blue) as judged by Hoechst staining. B, cell cycle
distribution of GM08505 (upper panel) and PSNF5
(lower panel) cells as determined by flow
cytometry. C, indirect immunofluorescence of hRAD51
(green) and incorporated BrdUrd (red) in GM08505
(upper panel) and PSNF5 (lower
panel) cells. Co-localization of signals is seen as
yellow.
View larger version (29K):
[in a new window]
Fig. 6.
Interaction between Sgs1 and Rad51.
A, interaction in the yeast two-hybrid system between Rad51
and various fragments of Sgs1 as indicated. Also shown are three
negative controls: MAX and HM12, which do not interact with Rad51, and
an empty vector control, demonstrating that the shortest Sgs1 fragment
that interacts with Rad51 does not alone activate expression of
-galactosidase from the reporter plasmid. B, growth on
YPD plates of sgs1
, rad51
, and
sgs1
rad51
mutants compared with wild type
cells in the absence or presence of 15 mM HU or 0.002% MMS
as indicated above. C, growth inhibitory effect of HU or
MMS, as indicated, on sgs1
(
), rad51
(
), and sgs1
rad51
(
) mutants
compared with wild type cells (
), as judged by cell number.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
srs2
mutant. One
interpretation of these data is that RecQ helicases act downstream of
RAD51, which is consistent with the known enzymatic properties of these
two classes of proteins. RAD51 catalyzes the early steps of HR, namely
the pairing of homologous sequences and exchange of DNA strands to form
a Holliday junction recombination intermediate (22, 24). Several RecQ
helicases, including BLM and Sgs1, can bind to and disrupt such
recombination intermediates (17-20). More specifically, we have shown
previously that BLM can promote branch migration of Holliday junctions
(19). Such an activity could either promote the formation of extensive stretches of heteroduplex DNA or conversely "destroy" recombination intermediates depending on the direction in which the Holliday junction
is translocated. Dual, but opposing, effects on RecA-mediated joint
molecule formation have been reported to be a feature of the E. coli RecQ protein (17). The interaction between hRAD51 and BLM may
therefore serve to recruit BLM to sites of recombinational repair. If
the role of BLM is to disrupt recombination intermediates by catalyzing
reverse branch migration, then loss of BLM would give rise to excessive
recombination. Conversely, if the normal role of BLM is to promote
branch migration, then the absence of BLM may lead to incomplete or
inappropriate processing of Holliday junctions. Such events could give
rise to the cytogenetic abnormalities seen in BS cells. It will
therefore be of interest to determine how directionality, if any, is
imposed on the branch migration activity catalyzed by BLM in
vivo. One possibility is that hRAD51 may load BLM onto Holliday
junctions in a particular orientation that would then dictate the
direction of junction translocation.
cells do not exhibit
hypersensitivity to ionizing radiation (40). However, in diploid yeast,
where DNA DSBs may potentially be repaired using either the sister
chromatid or the homologous chromosome, deletion of SGS1
confers x-ray sensitivity (40), implicating Sgs1 in the HR pathway for
repair of certain DNA DSBs.
View larger version (14K):
[in a new window]
Fig. 7.
Potential role for BLM in the
re-establishment of replication following replication fork collapse at
a DSB. See text for details.
In summary, BS cells show DNA recombination defects, and here we have
established an evolutionarily conserved molecular association between
RecQ helicases and Rad51 that links BLM with the pathway of HR. Further
studies using purified BLM and hRAD51, in addition to other components
of the recombinational repair pathway, will, we hope, shed light on the
role that BLM plays during this conserved process, and how aberrant
recombination can lead to destabilization of the genome and ultimately tumorigenesis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank members of the Imperial Cancer Research Fund Genome Integrity Group for valuable discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Imperial Cancer Research Fund (to L. W., S. L. D., and I. D. H.) and by a grant from the Medical Research Council (to N. C. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-1865-222427;
Fax: 44-1865-222431; E-mail: wu@icrf.icnet.uk.
Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M009471200
2 P. S. North, et al., manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: BS, Bloom's syndrome; GST, glutathione S-transferase; HU, hydroxyurea; HR, homologous recombination; MMS, methyl methanesulfonate; SCE, sister chromatid exchange; DSB, double-strand break; PCR, polymerase chain reaction; BrdUrd, bromodeoxyuridine.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | German, J. (1993) Medicine 72, 393-406[Medline] [Order article via Infotrieve] |
2. | Ellis, N. A., Groden, J., Ye, T. Z., Straughen, J., Lennon, D. J., Ciocci, S., Proytcheva, M., and German, J. (1995) Cell 83, 655-666[Medline] [Order article via Infotrieve] |
3. | Karow, J. K., Wu, L., and Hickson, I. D. (2000) Curr. Opin. Genet. Dev. 10, 32-38[CrossRef][Medline] [Order article via Infotrieve] |
4. | Yu, C., Oshima, J., Fu, Y., Wijsman, E. M., Hisama, F., Alisch, R., Matthews, S., Najura, J., Miki, T., Ouais, S., Martin, G. M., Mulligan, J., and Schellenberg, G. D. (1996) Science 272, 258-262[Abstract] |
5. | Kitao, S., Shimamoto, A., Goto, M., Miller, R. W., Smithson, W. A., Lindor, N. M., and Furuichi, Y. (1999) Nat. Genet. 22, 82-84[CrossRef][Medline] [Order article via Infotrieve] |
6. | Shen, J. C., and Loeb, L. A. (2000) Trends Genet. 16, 213-220[CrossRef][Medline] [Order article via Infotrieve] |
7. | Vennos, E. M., and James, W. D. (1995) Dermatol. Clin. 13, 143-150[Medline] [Order article via Infotrieve] |
8. | Huang, S., Li, B., Gray, M. D., Oshima, J., Mian, I. S., and Campisi, J. (1998) Nat. Genet. 20, 114-116[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Shen, J. C.,
Gray, M. D.,
Oshima, J.,
Kamath-Loeb, A. S.,
Fry, M.,
and Loeb, L. A.
(1998)
J. Biol. Chem.
273,
34139-34144 |
10. |
Wu, L.,
Davies, S. L.,
North, P. S.,
Goulaouic, H.,
Riou, J. F.,
Turley, H.,
Gatter, K. C.,
and Hickson, I. D.
(2000)
J. Biol. Chem.
275,
9636-9644 |
11. | German, J., Crippa, L. P., and Bloom, D. (1974) Chromosoma 48, 361-366[Medline] [Order article via Infotrieve] |
12. |
Sonoda, E.,
Sasaki, M. S.,
Morrison, C.,
Yamaguchi-Iwai, Y.,
Takata, M.,
and Takeda, S.
(1999)
Mol. Cell. Biol.
19,
5166-5169 |
13. |
Wang, W.,
Seki, M.,
Narita, Y.,
Sonoda, E.,
Takeda, S.,
Yamada, K.,
Masuko, T.,
Katada, T.,
and Enomoto, T.
(2000)
EMBO J.
19,
3428-3435 |
14. | Gangloff, S., McDonald, J. P., Bendixen, C., Arthur, L., and Rothstein, R. (1994) Mol. Cell. Biol. 14, 8391-8398[Abstract] |
15. |
Watt, P. M.,
Hickson, I. D.,
Borts, R. H.,
and Louis, E. J.
(1996)
Genetics
144,
935-945 |
16. |
Stewart, E.,
Chapman, C. R.,
Al-Khodairy, F.,
Carr, A. M.,
and Enoch, T.
(1997)
EMBO J.
16,
2682-2692 |
17. |
Harmon, F. G.,
and Kowalczykowski, S. C.
(1998)
Genes Dev.
12,
1134-1144 |
18. | Bennett, R. J., Keck, J. L., and Wang, J. C. (1999) J. Mol. Biol. 289, 235-248[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Karow, J. K.,
Constantinou, A.,
Li, J.-L.,
West, S. C.,
and Hickson, I. D.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
6504-6508 |
20. |
Constantinou, A.,
Tarsounas, M.,
Karow, J. K.,
Brosh, R. M.,
Bohr, V. A.,
Hickson, I. D.,
and West, S. C.
(2000)
EMBO Rep.
1,
80-84 |
21. | Ogawa, T., Yu, X., Shinohara, A., and Egelman, E. H. (1993) Science 259, 1896-1899[Medline] [Order article via Infotrieve] |
22. | Sung, P. (1994) Science 265, 1241-1243[Medline] [Order article via Infotrieve] |
23. | Benson, F. E., Stasiak, A., and West, S. C. (1994) EMBO J. 13, 5764-5771[Abstract] |
24. | Baumann, P., Benson, F. E., and West, S. C. (1996) Cell 87, 757-766[Medline] [Order article via Infotrieve] |
25. |
Paques, F.,
and Haber, J. E.
(1999)
Microbiol. Mol. Biol. Rev.
63,
349-404 |
26. | Lim, D. S., and Hasty, P. (1996) Mol. Cell. Biol. 16, 7133-7143[Abstract] |
27. |
Tsuzuki, T.,
Fujii, Y.,
Sakumi, K.,
Tominaga, Y.,
Nakao, K.,
Sekiguchi, M.,
Matsushiro, A.,
Yoshimura, Y.,
and Morita, T.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
6236-62340 |
28. |
Sonoda, E.,
Sasaki, M. S.,
Buerstedde, J. M.,
Bezzubova, O.,
Shinohara, A.,
Ogawa, H.,
Takata, M.,
Yamaguchi-Iwai, Y.,
and Takeda, S.
(1998)
EMBO J.
17,
598-608 |
29. | Haaf, T., Golub, E. I., Reddy, G., Radding, C. M., and Ward, D. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2298-2302[Abstract] |
30. | Zervos, A. S., Gyuris, J., and Brent, R. (1993) Cell 72, 223-232[Medline] [Order article via Infotrieve] |
31. |
Karow, J. K.,
Chakraverty, R. K.,
and Hickson, I. D.
(1997)
J. Biol. Chem.
272,
30611-30614 |
32. | Gietz, D., St. Jean, A., Woods, R. A., and Schiestl, R. H. (1992) Nucleic Acids Res. 20, 1425[Medline] [Order article via Infotrieve] |
33. |
Ishov, A. M.,
Sotnikov, A. G.,
Negorev, D.,
Vladimirova, O. V.,
Neff, N.,
Kamitani, T.,
Yeh, E. T. H.,
Strauss, J. F., III,
and Maul, G. C.
(1999)
J. Cell Biol.
147,
221-233 |
34. | Zhong, S., Hu, P., Ye, T.-Z., Stan, R., Ellis, N. A., and Pandolfi, P. P. (1999) Oncogene 18, 7941-7947[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Yankiwski, V.,
Marciniak, R. A.,
Guarente, L.,
and Neff, N. F.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5214-5219 |
36. | Dutertre, S., Ababou, M., Onclercq, R., Delic, J., Chatton, B., Jaulin, C., and Amor-Gueret, M. (2000) Oncogene 19, 2731-2738[CrossRef][Medline] [Order article via Infotrieve] |
37. | Tashiro, S., Kotomura, N., Shinohara, A., Tanaka, K., Ueda, K., and Kamada, N. (1996) Oncogene 12, 2165-2170[Medline] [Order article via Infotrieve] |
38. |
Yamagata, K.,
Kato, J.,
Shimamoto, A.,
Goto, M.,
Furuichi, Y.,
and Ikeda, H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8733-8738 |
39. |
Mullen, J. R.,
Kaliraman, V.,
and Brill, S. J.
(2000)
Genetics
154,
1101-1114 |
40. | Gangloff, S., Soustelle, C., and Fabre, F. (2000) Nat. Genet. 25, 192-194[CrossRef][Medline] [Order article via Infotrieve] |
41. | Hirschi, M., Netrawali, M. S., Remsen, J. F., and Cerutti, P. A. (1981) Cancer Res. 41, 2003-2007[Abstract] |
42. | Lu, X., and Lane, D. P. (1993) Cell 75, 765-778[Medline] [Order article via Infotrieve] |
43. | Wu, L., Davies, S. L., and Hickson, I. D. (2001) Cold Spring Harbor Symp. Quant. Biol., in press |
44. | Cox, M. M., Goodman, M. F., Kreuzer, K. N., Sherratt, D. J., Sandler, S. J., and Marians, K. J. (2000) Nature 404, 37-41[CrossRef][Medline] [Order article via Infotrieve] |
45. | Kowalczykowski, S. C. (2000) Trends Biochem. Sci. 25, 156-165[CrossRef][Medline] [Order article via Infotrieve] |
46. | Marians, K. J. (2000) Trends Biochem. Sci. 25, 185-189[CrossRef][Medline] [Order article via Infotrieve] |