From the Department of Biochemistry and Molecular
Biology, University of Texas Health Sciences Center, Houston, Texas
77030, the
Program in Cancer Biology, University of Texas
M. D. Anderson Cancer Center, Houston, Texas 77030, and the
§ Howard Hughes Medical Institute and Division of Biology,
California Institute of Technology, Pasadena, California 91125
Received for publication, August 22, 2000, and in revised form, October 6, 2000
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ABSTRACT |
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In a screen designed to discover suppressors of
mitotic catastrophe, we identified the Xenopus ortholog of
53BP1 (X53BP1), a BRCT protein previously identified in humans through
its ability to bind the p53 tumor suppressor. X53BP1 transcripts are
highly expressed in ovaries, and the protein interacts with Xp53
throughout the cell cycle in embryonic extracts. However, no
interaction between X53BP1 and Xp53 can be detected in somatic cells,
suggesting that the association between the two proteins may be
developmentally regulated. X53BP1 is modified via phosphorylation in a
DNA damage-dependent manner that correlates with the
dispersal of X53BP1 into multiple foci throughout the nucleus in
somatic cells. Thus, X53BP1 can be classified as a novel participant in
the DNA damage response pathway. We demonstrate that X53BP1 and its
human ortholog can serve as good substrates in vitro as
well as in vivo for the ATM kinase. Collectively, our
results reveal that 53BP1 plays an important role in the checkpoint
response to DNA damage, possibly in collaboration with ATM.
The eukaryotic cell cycle is a series of molecular events
resulting in the accurate replication and segregation of the genome. To
achieve this, it is essential that the successive phases of the cell
cycle are coordinated in a precise and punctual manner, since failure
to do so may result in abnormal growth and development, probably as a
consequence of genomic instability. For this purpose, cells have
evolved an elaborate network of "checkpoint controls" that monitor
the proper completion of various events in the cycle including DNA
replication in S phase and chromosome segregation during mitosis
(reviewed in Ref. 1). Upon genetic damage by any of a number of
mechanisms (UV light, ionizing radiation ( A growing body of information on the mechanisms of checkpoint control
in organisms ranging from yeast to humans have shown the importance of
these pathways with respect to maintaining genomic stability (1, 2).
For example, patients with the recessive disorder ataxia telangiectasia
(AT)1 fail to delay DNA
synthesis and mitosis in response to The biological response to DNA damage is best understood in both the
budding and fission yeast systems (1, 24). In many respects, the
general framework of the pathways responsible for checkpoint signaling
appears to be conserved from yeasts to humans. Here, DNA damage-sensing
proteins (e.g. various Rad gene products) transduce signals
to phosphoinositide (PI)-kinase like proteins (Mec1/Tel1 in budding
yeast, ATM and ATR in higher eukaryotes) that regulate effector kinases
and/or transcription factors (Rad53/Dun1 in budding yeast, Cds1/Chk2 in
higher eukaryotes; Refs. 9, 23, and 25-30). In Saccharomyces
cerevisiae, DNA damage slows S phase progression and can arrest
cells at either the G1/S or the G2/M
transitions (31, 32). In particular, Rad9 has been shown to be a
critical factor for various checkpoint responses (31-34). In addition
to Rad9, several other budding yeast gene products have been identified
to play important roles in sensing DNA damage or blocks in DNA
replication. These include Rad53, Mec3, Rad1, Rad24, Pol2, Dpb11, and
Rfc5 and their orthologous counterparts in other species (1, 25, 35,
36). Although the precise biochemical functions for many of these
proteins remain unknown, some DNA damage-sensing factors from various
systems appear to possess similarities to DNA polymerase
accessory factors, repair proteins, and exonucleases (25, 37-40). Such
properties might be expected for proteins whose functions are to sense
DNA damage.
One major checkpoint response in higher eukaryotes is activated upon
exposure to One protein that interacts with p53 is 53BP1 (48). 53BP1 was initially
identified in a two-hybrid screen with p53 as bait (48). Using murine
p53 amino acids (aa) 73-390 fused to the Gal4 DNA-binding domain, two
human proteins were identified which bound to p53. These proteins,
designated as 53BP1 and 53BP2, bound to the transcriptional activation
domain of p53 but not to a version of p53 with a point mutation (R175H)
in this domain (48). R175H generates a transcriptionally incompetent
p53 protein and has been found in patients with cancer (45), suggesting
that the binding of 53BP1 and 53BP2 is sensitive to the conformation of p53. Additional studies have shown that 53BP1 can enhance p53-mediated transcription of reporter genes (58). 53BP1 possesses strong homology
to the C-terminal repeats of BRCA1 (BRCT motifs). BRCT domains are
autonomously folding modules consisting of ~100 aa and are believed
to mediate protein-protein interactions, particularly with respect to
proteins associated with the cell cycle and the various aspects of DNA
metabolism: replication, recombination, and repair (49-51). Other
proteins possessing BRCT motifs include budding yeast Rad9 and Dpb11,
fission yeast Crb2 and Cut5, and mammalian XRCC1 and DNA ligase IV (1,
51, 52, 53). The C-terminal 271 aa of human 53BP1, the region
encompassing the BRCT folds, was found to interact with p53 through its
C-terminal BRCT repeats (48). Murine 53BP1 has also been isolated in
the two-hybrid assay when the inteferon-inducible p202 protein is used
as bait (54).
In this report, we present the isolation and characterization of the
Xenopus ortholog of human 53BP1 (X53BP1). We discovered X53BP1 in a screen designed to identify suppressors of mitotic catastrophe (negative regulators of the cell cycle) of the fission yeast Schizosaccharomyces pombe strain SP984 (55). We have
previously exploited the mitotic catastrophe phenotype of SP984 to
isolate Xorc2, a component of the origin recognition complex (56). This strain contains a temperature-sensitive mutation in wee1 and
a null mutation in the partially redundant mik1 gene. Both
genes encode proteins that modify and negatively regulate the Cdc2
kinase (Cdk1), a Cdk that functions in the progression into mitosis
(57). At the nonpermissive temperature of 35.5 °C, SP984 prematurely enters mitosis due to its inability to control the precocious activation of Cdc2. Thus, cDNA molecules capable of rescuing SP984 will express proteins that either directly or indirectly inhibit Cdc2
activity. The complete cDNA encoding X53BP1 presented here, in
conjunction with the recently described human sequence (58), reveals
that 53BP1 proteins from both frogs and humans are well conserved and
contain numerous putative ATM family member phosphorylation sites (59)
in addition to their BRCT motifs. Northern blotting reveals that X53BP1
expression appears largely restricted to ovaries. Consequently, we find
that the protein is abundant in eggs but much less so in somatic cells.
We present evidence that X53BP1 and Xp53 associate throughout the cell
cycle in a cell-free system, confirming the original two-hybrid
isolation of 53BP1 (48) in a specialized cellular setting. In contrast,
no association between X53BP1 and Xp53 can be detected in somatic
cells, suggesting that the interaction between these proteins is under
developmental control or is restricted to germline tissue.
Additionally, we find that X53BP1 is phosphorylated in vivo
in response to DNA damage. Consistent with this, we demonstrate that
both the frog and human 53BP1 proteins serve as substrates for the ATM
kinase in vitro, particularly in an N-terminal region
containing numerous ATM phosphorylation motifs. In addition, ATM
phosphorylates human 53BP1 in an N-terminal region in vivo
in response to Isolation of the Gene Encoding X53BP1 as a Suppressor of Mitotic
Catastrophe and Cloning of the Full-length Gene--
The fission yeast
strain SP984 (55) was transformed with a Xenopus laevis
cDNA library prepared in the S. pombe expression vector
pAX-NMT (56, 60) by the spheroplast method using Lipofectin (56).
pAX-NMT is a fission yeast/Escherichia coli shuttle vector and contains the S. cerevisiae LEU2 gene for selection. From
~200,000 transformants, about 60 colonies grew at the nonpermissive
temperature of 35.5 °C on minimal media lacking leucine and
thiamine. One rescuant, pC535, was chosen for further study. pC535
plasmid DNA was isolated and used to re-rescue SP984 from mitotic
catastrophe at the nonpermissive temperature. Once the rescuing event
was confirmed, the 1.9-kb ApaI/XhoI cDNA
insert of pC535 was used to probe a Xenopus oocyte library
as described previously (56, 60). This screen yielded S48-1, which
contains a 5.6-kb cDNA insert corresponding to X53BP1. To isolate
the 5'-end of X53BP1, sequence information at the 5'-end of S48-1 was
used to perform RACE PCR as described below. DNA sequencing of the
cDNA insert of S48-1 was done by primer walking with custom
designed oligonucleotides. The complete cDNA sequence for X53BP1
was assembled with information generated from S48-1 and pZX-1 (see below).
Total RNA Extraction, 5'-RACE PCR, and Northern
Hybridization--
Various tissues as described under
"Results" were collected from adult female X. laevis frogs and immediately frozen in liquid nitrogen. Total RNA
was extracted from the various tissues or from somatic
Xenopus tissue culture (XTC) cells using Trizol (Life Technologies, Inc.) in conjunction with DNase I. 5'-RACE PCR was performed with a RACE system (Life Technologies, Inc.) according to the
manufacturer's instructions. First strand cDNA synthesis was
synthesized from 1.0 µg of total XTC RNA using the GSP1 primer: 5'-AAAGAAGAATCCTTAG-3'. After dC tailing, a PCR was carried out using
the cDNA as a template along with an abridged anchor primer and the
nested GSP2 primer: 5'-CAGCATCTTGGGAAGGAGAAACCAGAT-3'. A dilution of
the original PCR was reamplified with a nested GSP3 primer:
5'-CAGAAGGATGGAGTAGGCGCAAACTGT-3'. All reactions were carried out under
the recommended conditions of the supplier. PCR products were
electrophoresed on 1.0% agarose gels, and a target band of ~0.9 kb
was gel purified and ligated into the pGEM-T vector (Promega), creating
pZX-1. DNA sequencing of pZX-1 was performed on both strands by primer
walking using an ABI sequencer and shown to unambiguously overlap with
S48-1.
For Northern hybridization, 10 µg of total RNA was separated on a
1.0% formaldehyde agarose gel with a 0.4-9.5-kb RNA ladder (Life
Technologies, Inc.). RNA was transferred onto Hybond-N+ membrane
(Amersham Pharmacia Biotech) by capillary elution with 20× SSC and
fixed with 0.5 N sodium hydroxide. The membrane was prehybridized at 65 °C for 15 min in rapid hybridization buffer (Amersham Pharmacia Biotech) and hybridized with a 0.9-kb probe corresponding to the extreme 5'-end of X53BP1 or a 1.2-kb fragment derived from Xenopus EF-1 Cell-free Extracts--
Xenopus cell-free
extracts were prepared by the method of Murray (53). Interphase
extracts were generated by the addition of 0.4 mM calcium
chloride to cytostatic factor (CSF)-arrested extracts in the presence
of cycloheximide (100 µg/ml) and allowed to proceed for 30 min at
25 °C. The entry into interphase was verified through either H1
kinase assays or observation of nuclear envelope formation.
Recombinant Proteins and Antibody Production--
To produce
polyclonal rabbit antibodies against X53BP1, we generated a GST fusion
protein between GST and residues 1652-2055 of X53BP1 by PCR
amplification with a high fidelity DNA polymerase, creating
GST-XBP1-5. This amplified X53BP1 fragment was ligated in frame
into pGEX4T-1 (Amersham Pharmacia Biotech) on a BamHI fragment. The resulting recombinant was confirmed and used to transform
DH10B cells. Protein expression was induced by the addition of 0.1 mM isopropyl-1-thio- DNA Damage-inducible Phosphorylation of X53BP1--
XTC cells
were cultured in Liebovitz' L-15 medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum and antibiotics. 36 plates were
grown to 60% confluency. One half of the plates was treated with 10 Gy
of Indirect Immunofluorescence in XTC Cells--
XTC cells were
grown on polylysine-treated coverslips to 60% confluency in
Liebovitz' L-15 medium and processed for indirect immunofluorescence
with ATM Kinase Assays--
In vitro ATM kinase assays
were performed essentially as described previously (7, 8). Briefly,
293T cells were transfected with constructs expressing either the
full-length ATM gene or a kinase-defective version of the enzyme. The
expressed proteins were immunoisolated with M2/M5 FLAG antibodies
(Sigma) coupled to protein A/G-Sepharose (Santa Cruz Biotechnology,
Inc., Santa Cruz, CA) and incubated with candidate GST fusion proteins
containing various segments of either the Xenopus or human
53BP1 proteins. Approximately 1 µg of affinity-purified, candidate
substrate protein was incubated in ATM kinase buffer (8) with 10 µCi
of [ Identification of X53BP1 as a Suppressor of Mitotic
Catastrophe--
To identify potential negative regulators of the cell
cycle, we rescued the fission yeast mitotic catastrophe strain SP984 (55) with a Xenopus cDNA library prepared in the pAX-NMT
S. pombe expression vector (56, 60). At the nonpermissive
temperature of 35.5 °C, SP984 is deficient in Cdc2-specific tyrosine
kinase activity, an inhibitory modification. This results in a
precocious activation of Cdc2 during interphase, causing premature
mitotic entry and cell death. We have previously used this method to
identify Xorc2, a member of the Xenopus origin recognition
complex (56). A second rescuant described here, pC535 (Fig.
1), was found to contain a 1.9 kb
ApaI/XhoI cDNA fragment encoding for the
C-terminal region of the frog 53BP1 ortholog (X53BP1). 53BP1 was
originally described as a human protein capable of binding to the
central transactivation domain of murine p53 in a two-hybrid assay
(48). The largest X53BP1 plasmid capable of rescuing SP984 was found to
contain a 4-kb ApaI/XhoI fragment encoding for
the C-terminal region of X53BP1. All rescuing cDNAs of X53BP1
possess the two repeating BRCT motifs found at the extreme C terminus
(Fig. 2A). pC535-expressing
mutant cells divided normally and exhibited neither an obvious
wee nor elongated phenotype (not shown). Despite
this, the rescued cells still entered mitosis inappropriately in the presence of 10 mM hydroxyurea at the restrictive
temperature. Thus, despite its formal classification as a negative
regulator of the cell cycle in fission yeast, the expression of X53BP1
is not sufficient to restore the replication checkpoint defect
characteristic of SP984. The precise reasons for why X53BP1 rescues
SP984 from mitotic catastrophe remain to be determined.
Cloning and Characterization of X53BP1--
Because human 53BP1 is
comprised of 1972 amino acids (58), we assumed that the frog ortholog
would be of a similar size. If so, then it was clear that rescuant
pC535 was only a partial cDNA clone. In an attempt to isolate the
full coding sequence of X53BP1, we used the 1.9-kb
ApaI/XhoI fragment from pC535 to screen a
cDNA library derived from X. laevis oocytes (60). From 1 million library clones, 22 cDNAs encoding various portions of X53BP1 were isolated. The largest isolate, S48-1, was sequenced and
found to be an incomplete cDNA, since it contained a 5.6-kb ApaI/XhoI fragment. To isolate the 5'-end of
X53BP1, we performed a PCR RACE experiment (Life Technologies, Inc.)
with RNA isolated from the XTC somatic cell line using sequences
derived from the 5'-end of S48-1. A 0.9-kb fragment was successfully
isolated, sequenced on both DNA strands, and shown to encode the
N-terminal region of X53BP1. Importantly, this RACE product possesses
an initiating methionine codon that properly aligns with the proposed first methionine of human 53BP1 (Ref. 58; Fig. 2A). This
methionine residue is also preceded by an in-frame stop codon (UAG) six
nucleotides upstream of the ATG codon (not shown). Moreover, this
fragment contains a significant and unambiguous 3' overlap to the
5'-end of S48-1 (not shown). The full-length cDNA for X53BP1 is
~6.5 kb long and contains 5'- and 3'-untranslated regions of 45 and 62 nucleotides (excluding the poly(A) tail), respectively. The complete
sequence of X53BP1 encodes a protein of 2104 amino acids (predicted
mass of 231.3 kDa) with significant homology to the 1972-residue
human 53BP1 protein and has been deposited in GenBankTM
(accession number AF281071).
Both 53BP1 proteins possess two repeating BRCT motifs (49, 50, 61) at
their respective C termini. Additionally, the frog and human 53BP1
orthologs contain numerous "(S/T)Q" motifs, some of which
are conserved, particularly in their N termini, where there is
clustering (Fig. 2A). Such sequence elements have been shown
to formulate strong recognition determinants for the ATM kinase and its
related family members ATR and DNA-PK (59). In addition, X53BP1
contains four (S/T)PX(R/K) motifs that reside in potential
Cdk recognition sites (not shown). Thus, both the frog and human 53BP1
proteins possess sequence features suggestive of functions in
checkpoint control during the biological response to DNA damage.
To further understand the role of X53BP1 in Xenopus, we
analyzed X53BP1 transcripts by Northern blotting (Fig. 2B).
10 µg of total RNA was isolated from various female frog tissues with Trizol and electrophoresed on 0.8% agarose gels. The gels were processed for Northern analysis with a 0.9-kb DNA probe specific for
the 5'-end of X53BP1. As a loading control, a 1.1-kb PstI probe specific for Xenopus EF-1 Association of X53BP1 and Xp53 in Cell-free Extracts and Somatic
Cells--
To characterize X53BP1 further, we raised polyclonal
antibodies that react against a glutathione S-transferase
(GST) fusion protein encoding amino acids 1652-2055 (GST-XBP1-5),
which reside near the C terminus of X53BP1. The soluble GST-XBP1-5
protein was purified on glutathione-agarose (Sigma), eluted with
reduced glutathione as described (64), and injected into rabbits for polyclonal antibody production. Affinity-purified antibodies that react
against X53BP1 (
Because 53BP1 was initially identified as a p53-binding protein, we
investigated the relationship between X53BP1 and the Xenopus p53 protein (Xp53; Refs. 65-67) in cell-free extracts derived from frog eggs. Xp53 is highly expressed during oocyte development (68).
Xenopus eggs contain an abundance of Xp53 protein, which has
been shown to possess the same biochemical properties of its human
counterpart (63, 66, 69). Despite this, there is no transcription
occurring in eggs, since development relies upon maternal stores of
protein until zygotic transcription occurs at the midblastula
transition (70). Thus, the functional significance of Xp53 in early
embryonic development is not clear, although roles for the protein
during this period have been suggested. For example, microinjection of
Xp53 mRNA into early cleavage stages interferes with normal frog
development (67). In addition, inhibition of Xp53 function via ectopic
expression of either a dominant negative Xp53 or its inhibitor X-dm2
results in an early block to differentiation (71).
To address the nature of any X53BP1/Xp53 interactions, we performed
reciprocal coimmunoprecipitation experiments (Fig. 3B) from
cell-free extracts derived from unfertilized eggs. For this purpose, we
generated polyclonal antibodies against the frog p53 protein (
To further characterize the interaction between X53BP1 and Xp53, we
performed reciprical coimmunoprecipitations in somatic XTC cells
extracts. Immunoprecipitations from these extracts were processed in
the same manner as described for the egg extracts. Unexpectedly, no
interaction can be detected between X53BP1 and Xp53 in somatic cells
(Fig. 3D). Because p53 is known to be involved in the
response to DNA Damage-inducible Phosphorylation of X53BP1--
Many proteins
involved in checkpoint signaling are modified in response to DNA
damage. These include the ATM-dependent phosphorylation of
BRCA1, Cds1/Chk2, p53, and p95/Nbs1 (8, 9, 11-14). Given the
relationship of 53BP1 to p53, the presence of its two-repeating BRCT domains, and its potential ATM phosphorylation sites, X53BP1 appeared as a strong candidate for modification by phosphorylation in
response to DNA damage, particularly Subcellular Localization of X53BP1 in Somatic Cells by Indirect
Immunofluorescence--
Previous indirect immunofluorescence (IF)
experiments with human 53BP1 revealed that the protein can be found in
both the cytoplasm and nucleus (58). The nuclear immunostaining
observed for 53BP1 in these studies revealed that the protein could be found in a homogenous or punctate pattern. However, because these studies were done with cells that had been transfected with 53BP1, the
subcellular localization of the endogenous protein could not be
unambiguously determined. We used IF to determine the subcellular localization of the endogenous frog X53BP1 protein in XTC cells. Asynchronous cells were grown to 60% confluency on polylysine-coated coverslips and processed for IF analysis with affinity-purified ATM-dependent Phosphorylation of 53BP1--
The
sequences of both X53BP1 and 53BP1 from humans suggest that the
proteins may serve as effective substrates for members of the ATM
kinase family (e.g. ATM, ATR, and DNA-PK). Both the frog and
human 53BP1 proteins possess numerous (S/T)Q motifs, which are known to
be the minimal, essential determinants for recognition by ATM family
members (59). X53BP1 possesses 32 of these potential recognition
motifs, whereas the human ortholog has 30 such sites. Several of these
sites are clustered in the N-terminal region of X53BP1 (Fig.
2A). Such clustering is also evident in the N-terminal
region of human 53BP1 (Ref. 58; Fig. 2A). Clustered (S/T)Q
motifs have been suggested to formulate a strong recognition
determinant for ATM and its related PI-like kinases (11, 59). Given
these potential phosphorylation sites and the fact that X53BP1 is
phosphorylated in response to
Our results demonstrate that GST fragment 1 (GST-XBP1-1) and
GST-XBP1-4 serve as good in vitro substrates for ATM but
not the KD version of the protein (Fig. 4). On the other hand,
GST-XBP1-2, GST-XBP1-3, and GST-XBP1-5 appear to be essentially
refractory to ATM-dependent phosphorylation in this assay
despite the fact that these sequences contain several potential ATM
recognition sites. GST-XBP1-1 encodes for aa residues 1-338 in
X53BP1, a region of the protein that contains 11 putative ATM
phosphorylation sites (Fig. 2A). Interestingly, GST-XBP1-1
migrates around 100 kDa on 6% SDS gels despite the fact that its
predicted molecular mass is ~65 kDa. Such anomalous behavior in SDS
gels has also been observed for human 53BP1 (Ref. 58; Fig.
6). X53BP1 has a predicted mass of 231 kDa but migrates at ~300 kDa (Figs. 3A and 4).
Phosphorylation of GST-XBP1-1 by ATM appears to generate a doublet
pattern, suggesting that the enzyme targets multiple residues within
the first 338 amino acids of X53BP1 (Fig. 4). In contrast, we find that
ATM-dependent phosphorylation of GST-XBP1-4, a fusion
protein encoding X53BP1 residues 1201-1657, generates an apparent
singlet, despite the fact that there are eight (S/T)Q motifs within
this region. It is interesting to note that four of the potential
phosphorylation sites of GST-XBP1-4 reside in a cluster spanning
residues 1272-1327 (not shown). Taken together, these results clearly
demonstrate that X53BP1 is phosphorylated in vitro at
multiple places by the ATM kinase.
To further investigate the relationship between ATM phosphorylation and
53BP1, we created a GST fusion protein between human 53BP1 residues
1-524 (HBP1-524) and assayed for its potential to be recognized as a
substrate by the ATM kinase. Human 53BP1 possesses 11 potential ATM
kinase recognition motifs within this region of the protein (Ref. 58;
Fig. 2A). Additionally, HBP1-524, like its amphibian
counterpart, migrates much slower than its predicted molecular weight.
Thus, N-terminal sequences of both the frog and human 53BP1 proteins
contribute to the anomalous mobility that has been observed for these
proteins in SDS gels (58). Nevertheless, HBP1-524 serves as a very
good substrate for ATM in our in vitro assay and appears as
a phosphorylated doublet (Fig.
7A). Because the N-terminal
regions of both Xenopus and human 53BP1 proteins appear to
be phosphorylated by ATM in vitro, we assessed the ability
of the N-terminal region of human 53BP1 to serve as a potential
substrate for ATM in vivo. Human 53BP1 residues 1-524 were
cloned in frame into pFLAG-NLS lox (11), generating plasmid pZX-12.
This plasmid expresses residues 1-524 fused to an N-terminal nuclear
localization signal and to a FLAG epitope tag (FLAGBP1-524). As
previously seen for this segment of 53BP1, H53BP1-524 also ran
aberrantly on SDS gels. ATM-dependent phosphorylation was
assessed in a transient transfection assay using 293T cells. These
cells were transfected with pZX-12 alone or in combination with
plasmids encoding either the ATM WT or KD protein. After 48 h, the
cells were exposed to either 0 or 50 Gy of The accurate replication and segregation of the genetic material
is tightly controlled during the cell cycle. It has become increasingly
clear that cells that cannot properly coordinate these processes within
the framework of the cell cycle place themselves at great risks for
developing chromosomal instabilities. Consequently, the faithful
transmission of the genetic material will be compromised, leading to a
number of potential pathologies including cancer (2). Cells respond to
various forms of DNA damage by halting cell cycle progression and
activating the machinery responsible for mending disabled DNA (1, 31,
32). One form of DNA damage is DSBs, which occur spontaneously during S
phase, meiosis, and V(D)J recombination and through environmental
agents such as We identified X53BP1 in a genetic screen designed to uncover
suppressors of mitotic catastrophe. We observed that X53BP1-expressing cDNAs could rescue SP984 as effectively as wee1, the
natural complementing gene. X53BP1 cDNAs were found to rescue SP984
only when expressed under the highly inducible nmt1
promoter, implying that high levels of X53BP1 are responsible for the
rescued phenotype. Additionally, all rescuing cDNAs encoded for
X53BP1 derivatives possessing the C-terminal BRCT repeats. Thus, it is
possible that overexpression of these repeated BRCT motifs leads to the
titration of a cellular factor(s) that can either directly or
indirectly influence the activity of Cdc2 and hence the ability of
SP984 to undergo premature mitosis. Overexpression of X53BP1 in SP984,
however, is not sufficient to restore the replication checkpoint defect
associated with the strain as the rescued cells undergo a mitotic
catastrophe when exposed to hydroxyurea. It is therefore unlikely that
X53BP1 suppresses the growth defect of SP984 through the activation of
the replication checkpoint. Thus, the precise reasons for the ability
of X53BP1 to rescue SP984 remain unknown, although it is interesting to note that a yeast strain deficient in a checkpoint mechanism can be
rescued by a protein that itself appears to function in a checkpoint response to DNA damage. Whatever the case, it is clear that X53BP1, when expressed in SP984, influences the growth of the yeast. Perhaps this accounts for its fortuitous isolation. Interestingly, it has been
reported that the C-terminal BRCT repeats of BRCA1 can also confer
growth-suppressing properties in budding yeast (73). Although the
mechanism of this suppression is unknown, it was shown to require
intact BRCT motifs. Despite these observations, the transformation of
pC535 into wild type yeast cells does not impair the growth of the strain.
Unlike human 53BP1 transcripts, X53BP1 is highly expressed in ovarian
tissue as revealed by Northern blotting with Xenopus total
RNA. Consequently, X53BP1 is abundant in unfertilized eggs. The levels
of X53BP1 do not appear to be cell cycle-regulated during early
development as judged by immunoblotting of cell-free extracts staged at
either mitosis or interphase. We do observe, however, that the diffuse
banding pattern of X53BP1 in egg extracts is quite different from the
compact one seen for the somatic form of the protein. Whether this
reflects alternate forms of X53BP1 (i.e. isoforms or
posttranslationally modified forms) remains to be determined. Because
of the abundance of the protein, X53BP1 can be readily detected
throughout the cell cycle in complexes with Xp53 through reciprocal
coimmunoprecipitation experiments. It appears that a very large
fraction of the embryonic X53BP1 is associated with Xp53 as judged by
depletion analysis.3
Previously, it has been observed that many proteins involved in early
development are abundant in unfertilized eggs. This includes factors
responsible for the rapid cell divisions that take place in early
development, including DNA replication initiation proteins such as
Xorc2, Xcdc6, and RPA, as well as the Xp53 transcription factor (56,
63, 74, 75). We anticipate that the large maternal stores of X53BP1 and
Xp53 reflect a previously unappreciated role for these proteins during
development. During early Xenopus development, zygotic
transcription does not occur until the midblastula transition (70),
eliminating any role for Xp53 as a transcription factor during this
window of time. Checkpoint response mechanisms are not established
until after midblastula transition, since treatment of embryos staged
prior to this with DNA-damaging agents does not elicit a cell cycle
arrest (76, 81). Nevertheless, previous studies have implicated Xp53 in
frog development (67, 71). Based upon our findings that X53BP1 and Xp53
form complexes during this time period, it is possible that X53BP1 may
influence the Xenopus developmental program and/or the
biology of p53. Unexpectedly, we failed to observe an interaction
between X53BP1 and Xp53 in somatic XTC cells. Similar
observations have been made regarding 53BP1 and p53 in human cell
lines.2 Thus, for reasons presently unclear, the
association between X53BP1 and Xp53 appears developmentally regulated
or restricted to germline tissue.
Our findings establish X53BP1 as a protein that is phosphorylated in
response to DNA damage. In particular, we have demonstrated that
Immunofluorescence experiments in asynchronous XTC cells reveal that
X53BP1 is diffuse within the cytoplasm but present in the nucleus of a
subset of cells in the form of a few large foci. Upon exposure to
In some respects, the general framework for the biological response to
DNA damage appears conserved from fungal systems to human. DNA
damage-sensing proteins activate PI kinase-like enzymes that transduce
the damaged-DNA signal to either effector kinases and/or transcription
factors (1, 35). The end result is an attenuation in cell cycle
progression and the activation of the DNA repair apparatus. Thus, many
of the gene products identified through yeast genetics might also be
expected to have conserved orthologs in higher eukaryotes. This appears
to be the case for many of the DNA damage sensors (budding
yeast/fission yeast Mec3/Hus1, Ddc/Rad9, and Rad17/Rad1), PI
3-kinase-like transducers Mec1/Rad3, effector kinases Chk1/Chk1 and
Rad53/Cds1, and many aspects of the DNA repair machinery (1, 9, 25-30,
78, 80). Although budding yeast does not utilize p53-based mechanisms
for cell cycle arrest, it activates analogous transcription-coupled
repair processes through Rad53 and Dun1 (1). Given this, one might
wonder how 53BP1 fits into any such generalized scheme, since no
obvious orthologs of the protein appear in the yeasts. However, 53BP1 contains homology to previously identified yeast DNA damage response proteins only through its BRCT motifs. This most notably includes budding yeast Rad9 and Dbp11 and fission yeast Crb2 and Cut5 (1, 31,
52), all of which are thought of as upstream regulators in the DNA
damage response (1, 52). If 53BP1 can be thought of in a similar
manner, then it is conceivable that the protein is part of the sensing
mechanism for DSBs that is transduced to ATM and/or its related family
members. We have observed that X53BP1 becomes phosphorylated shortly
after DNA damage (within 5 min),3 consistent with the
protein functioning as part of the initial biological response to
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-IR), chemicals, etc.),
checkpoint response pathways become activated, leading to attenuation
in cell cycle progression and a mobilization of the DNA repair machinery.
-IR, suggesting that the gene
responsible for this pleiotropic disease, ATM (mutated in
ataxia telangiectasia; Ref. 3), plays a major role in coordinating the
checkpoint response (4). Indeed, one of the hallmarks of AT is a
predisposition to cancer, probably as a result of the failure to repair
damaged DNA (5). In addition to ATM, many tumors and cancer
predisposition syndromes have defects in checkpoint-related gene
products that are phosphorylated and controlled, at least in part, by
the ATM kinase. This includes BRCA1 (breast cancer gene 1), the
effector kinase Cds1/Chk2, p95/Nbs1, and p53 (6-14). BRCA1 has been
reported to function in the transcriptional-coupled repair of damaged
DNA, G2/M checkpoint control, and the repair of
double-stranded DNA breaks (DSBs; Refs. 15-18). In addition, BRCA1
resides in a large complex of proteins (BASC) dedicated to repairing
various forms of damaged DNA (19). This includes DNA mismatch repair
proteins, the BLM helicase, and the RAD50-MRE11-NBS1 complex that is
believed to function in the repair of DSBs (21, 22). Recent work by
Elledge and colleagues has suggested that mutations in the ATM/BRCA1
pathway may account for 10% of all cases of breast cancer (11).
-IR or other agents that create DSBs. The PI-kinase like
protein ATM responds to this type of genotoxic insult (41) in a manner
that is most likely dependent upon DNA damage-sensing proteins.
ATM-dependent phosphorylation of the p53 tumor suppressor
is known to mediate cell cycle arrest in response to DNA damage
(42-44). The gene encoding p53 is the most frequently mutated gene in
all cases of human cancer (45). p53 protein levels are activated and
stabilized in cells exposed to
-IR. Here, the protein can act as a
tetrameric transcription factor by inducing the expression of genes
involved with the DNA damage response (42, 44). The precise mechanisms
by which p53 is activated are not well understood and are likely to be cell type-specific (44). In other instances, p53 directs the apoptotic
response (45). p53 protein levels are tightly regulated by the Mdm2
protein, an E3 ubiquitin ligase that targets p53 for degradation by the
proteosome. It is believed that multiple modifications at the N and C
termini contribute to the activation of latent p53 (44, 45). Such
modifications include phosphorylation by ATM on serine residue 15 (7, 8) and phosphorylation of serine 20 by Cds1/Chk2 (46, 47). Both of
these modifications are believed to contribute to the overall stability
of p53, perhaps by interfering with its ability to be degraded by Mdm2.
-IR. Taken together, our data indicate that 53BP1
plays a significant role in the cellular response to DNA damage,
classifying it as an important checkpoint response protein.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(62) for 2 h at 65 °C
in rapid hybridization buffer. The membrane was washed for 15 min at
65 °C for 15 min in 2× SSC, 1× SSC, and 0.1× SSC with 0.1% SDS
and exposed to film.
-D-galactopyranoside at
25 °C for 3 h. Soluble GST-XBP1-5 protein was affinity
purified on glutathione-agarose. The purified protein was used as
antigen for injection into rabbits (Covance). All other 53BP1 fusion
proteins were generated in a similar manner. Various bleeds were tested
for antibody production via immunoblotting with a chemiluminescent
system (Amersham Pharmacia Biotech), and those that were positive were
used to affinity-purify
X53BP1. Anti-GST antibodies were selectively
removed from the serum by adsorption onto GST-loaded Sepharose beads.
The unbound antibodies against X53BP1 were affinity-purified as
described (72) with GST-XBP1-5 protein coupled to CNBr-Sepharose
(Amersham Pharmacia Biotech) at a concentration of 1.0 mg/ml as per the manufacturer's instructions. For the generation of antibodies against
Xp53, the full coding sequence to Xp53 was isolated from a
Xenopus cDNA library (60), amplified with a high
fidelity DNA polymerase, and ligated in frame into pGEX4T-1 for protein expression. Soluble GST-Xp53 was expressed in DH10B bacterial cells and
affinity-purified as described above. The isolated protein was used as
an antigen to generate polyclonal antibodies.
-IR with a cesium irradiator, and the other half was left
untreated. Samples were allowed to recover for 1 h post-treatment.
After the recovery period, the cells were collected, washed with
phosphate-buffered saline (PBS), and lysed on ice with PBS supplemented
with 1% TX-100, 1% Nonidet P-40, 2 mM CaCl2,
and a mixture of protease inhibitors. Clarified supernatants were
treated for 1 h with
X53BP1 (or
Xp53 in some cases) that had
been covalently coupled to protein A-Sepharose. The immunoprecipitates were subsequently washed four times with EB+ buffer (20 mM
-glycerol phosphate, 10 mM Hepes, 1% Nonidet
P-40, and 25 mM sodium fluoride, pH 7.5). The beads
were further washed twice in an EB+ buffer without detergent and sodium
fluoride. Finally, the samples were washed twice in
phosphatase
buffer (50 mM Tris, 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij 35, pH 7.5) and split in
half. One half was treated with phosphatase buffer and the other was supplemented with 1.0 unit of
-phosphatase (New England Biolabs), incubated at 30 °C for 30 min, and loaded onto Novex 3-8%
acrylamide gels.
X53BP1 as follows. Cells were fixed with 2% paraformaldehyde
in 66% phosphate-buffered saline, pH 7.4, and permeabilized with
0.05% saponine, 1.5 mM NH4Cl in
phosphate-buffered saline. Permeabilized cells were treated with
primary antibody for 1 h, washed three times in phosphate-buffered
saline without paraformaldehyde, and incubated with an anti-rabbit IgG
conjugated to fluorescein isothiocyanate for 1 h (Pierce). Mounted
coverslips were viewed under oil at 100× magnification and
photographed using an Olympus BX60 microscope equipped with a Spot
digital camera (Diagnostics Instruments) interfaced. Images were
transferred and processed into Adobe Photoshop 5.0.
-32P]ATP for 30 min at 30 °C with constant
agitation. In vivo phosphorylation of 53BP1 was measured in
a transient transfection assay using 293T cells. Transfections were
performed with Lipofectin (Life Technologies, Inc.). Human 53BP1
residues 1-524 were fused in frame to pFLAG-NLS lox (11), creating
plasmid pZX-12. pZX-12 encodes FLAGBP1-124. 2.5 µg of pZX-12 was
transfected alone or cotransfected along with an equal amount of
plasmid expressing either the wild type or kinase-dead version of ATM.
After 48 h posttransfection, the cells were treated with either 0 or 50 Gy of ionizing radiation using a cesium irradiator. After a 1-h
recovery, the cells were harvested, processed for
immunoprecipitation, electrophoresed on 6% SDS gels, and
transferred overnight in preparation for Western analysis with
anti-FLAG M2/M5 antibodies (Sigma). FLAGBP1-1524 was visualized by
Western blotting with
X53BP1 and the ECL detection system (Amersham
Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Rescuing of a mitotic catastrophe strain
(SP984, mik::ura4+, wee 1-50 leu1-32
ade6-210 ura4-D18 h+N) by an X. laevis cDNA encoding X53BP1. A,
schematic representation of the screen utilized to isolate pC535, a
fission yeast/E. coli shuttle vector (pAX-NMT) containing a
1.9-kb ApaI/XhoI cDNA corresponding to the
C-terminal region of X53BP1. See "Experimental Procedures"
for details. B, suppression of mitotic catastrophe by pC535.
SP984 was transformed with either the pAX-NMT vector only, rescuant
pC535, or the wild type wee1 gene cloned into pAX-NMT, grown
for 6 days at the restrictive temperature of 35.5 °C, and streaked
out on agar plates.
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Fig. 2.
Characterization of the gene encoding
X53BP1. A, amino acid alignment of the N termini of
Xenopus and human 53BP1 proteins. The frog and human 53BP1
proteins (GenBankTM accession numbers AF281071 and
AF078776, respectively) were compared via a BLAST program (NCBI) as
shown. Potential ATM family member phosphorylation sites (59) are
highlighted in black and are also represented by the
vertical hatched lines. B,
Northern blot of X53BP1 transcripts in various X. laevis
tissues. 10 µg of total RNA was isolated and processed for Northern
blotting analysis as described under "Experimental Procedures." The
major X53BP1 transcript of 6.5 kb is present in abundance in ovaries
and to a lesser extent in the kidney-derived XTC cell line. Prolonged
exposures also reveal a minor amount of transcript in the heart. A
second, minor transcript migrating around 4.5 kb is also present.
was used (not shown).
These messages have been shown to be relatively constant in
Xenopus. (62). Our results clearly indicate that X53BP1
transcripts appear to be abundant in ovaries but not in the other
tissues that were sampled (Fig. 2B). Unlike
Xenopus, the human 53BP1 transcripts were detected in all
tissues sampled (48). As is also the case for human 53BP1, two
transcripts can be detected (48). However, in contrast to the frog
messages of ~7.0 and 4.5 kb (Fig. 2A), the human 53BP1
messages are 11.0 and 7.0 kb (48). X53BP1 mRNA is detectable,
albeit at much reduced levels relative to the ovaries, in heart tissue
and the somatic XTC cell line. The high X53BP1 transcript levels in
ovarian tissue, as has also been demonstrated for Xp53 (63), are
consistent with the maternal stockpiling that is seen for many
proteins, including X53BP1 protein (see below).
X53BP1), but not against GST, were isolated as
described under "Experimental Procedures" and used to examine the
protein in cell-free extracts derived from Xenopus eggs
(Fig. 3A). Such extracts have
been shown to recapitulate the major events of the cell cycle including
DNA replication and mitosis (53, 56, 60). Xenopus eggs are
arrested in metaphase of meiosis II (M phase), a mitotic-like state,
through the action of CSF. X53BP1 is present in both M phase and
interphase extracts in equivalent amounts as revealed by Western
blotting (Fig. 3A). Like its human counterpart, X53BP1
migrates aberrantly on SDS gels (58). The predicted molecular mass of
X53BP1 is 231 kDa, but both the M phase and interphase forms appear to
migrate around 300 kDa (Fig. 3A). X53BP1 was often detected
as a diffuse set of bands (Fig. 3A), consistent with the
protein undergoing posttranslational modifications, possibly
phosphorylation, throughout the cell cycle. To demonstrate that X53BP1
is present in somatic cells, in addition to embryonic extracts, we
examined the protein in XTC cells, a somatic cell line (79). We
immunoprecipitated X53BP1 from XTC cells with
X53BP1 and examined
the presence of the protein by Western blotting. Our results show that
X53BP1 is present in the XTC cell line but at much reduced levels
relative to the embryonic protein (Fig. 3A). We estimate
that the concentration of X53BP1 in Xenopus eggs is 100 nM and less than 10 nM in somatic cells. Unlike
the diffuse banding pattern often seen for embryonic X53BP1, the
somatic protein species appears to migrate as a much sharper band in
6% SDS gels (Fig. 3A).
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Fig. 3.
Analysis of X53BP1 in cell-free extracts and
somatic XTC cells. A, immunoblotting of cell cycle
staged extracts or somatic XTC cells for X53BP1. 10µl (~500 µg of
protein) from either mitotic (CSF; lanes 1 and
2) or interphase extract (lane 3) was
immunoprecipitated with either a control IgG (lane
1) or X53BP1 (lanes 2 and
3), electrophoresed on 6% SDS gels, and immunoblotted with
affinity-purified
X53BP1. X53BP1 migrates at ~300 kDa. Lane
4, XTC extract (~5-10 mg of protein) was immunoprecipitated
with
X53BP1 and immunoblotted for X53BP1 with
X53BP1.
B, coimmunoprecipitation of Xp53 with
X53BP1 in cell-free
extracts. 50 µl of either CSF-arrested (M;
lanes 1 and 2) or interphase-arrested
(I; lanes 3 and 4) extracts
was immunoprecipitated with a control IgG (lanes
1 and 3) or
X53BP1 (lanes
2 and 4) and processed for immunoblotting for
Xp53 with either
Xp53 or in some cases the Xenopus p53
antibody 2274 (66). C, coimmunoprecipitation of X53BP1 with
Xp53. 50 µl of either CSF-arrested (M; lanes
1 and 2) or interphase-arrested (I;
lanes 3 and 4) extracts were
immunoprecipitated with affinity-purified
Xp53 or a control rabbit
IgG (lanes 1 and 3) and processed for
immunoblotting with
X53BP1 (lanes 2 and
4). Visualization of target antigens was conducted with a
chemiluminescent system (ECL; Amersham Pharmacia Biotech).
D, coimmunoprecipitation of X53BP1 and Xp53 in XTC cells.
XTC cells were treated with either 0 or 10 Gy of
-IR and allowed to
recover for 1 h prior to making extracts. XTC extracts were
immunoprecipitated with
Xp53 (lanes 1 and
2) or
X53BP1 (lanes 3 and
4) and immunoblotted for either X53BP1 or Xp53 as
shown.
Xp53).
Coimmunoprecipitation experiments were performed with either
X53BP1,
Xp53, or a control rabbit IgG. In some instances, we immunoblotted
for Xp53 with antibody 2674 (
2674), a polyclonal Xp53 antiserum
described previously (66). We found that
Xp53 and
2674 gave
similar results (not shown). For immunoprecipitations, all antibodies
were covalently linked to protein A beads with dimethylpimelimidate as
described previously (72). After a 1-h incubation at 4 °C in either
CSF-arrested or interphase extracts, the beads were collected by brief
centrifugation, thoroughly washed, and then resuspended in SDS sample
prior to loading on SDS gels. The results clearly demonstrate that
X53BP1 and Xp53 associate with each other throughout the cell cycle in
cell-free extracts (Fig. 3, B and C). Xp53 can be
detected in X53BP1 immunoprecipitates, and X53BP1 is present in Xp53
immunoprecipitates as shown. Neither of these two proteins are present
with the control IgG, indicating that the association between X53BP1
and Xp53 is specific. Thus, consistent with previous studies in mammals
(48, 58), X53BP1 and Xp53 form complexes in Xenopus.
Interestingly, such complexes are present throughout the cell cycle in
both M phase and interphase.
-IR DNA damage, we assayed for a potential X53BP1/Xp53
interaction in the presence of this form of genotoxic stress. As
observed before, no interaction between the two proteins can be seen.
Similar observations have also been made for 53BP1 and p53 in human
cell lines.2 Thus, these data
suggest that the association between X53BP1 and Xp53 is under
developmental control or is restricted to germline tissue.
-IR. To address this issue, we
examined the nature of X53BP1 in Xenopus somatic XTC cells
that had been exposed to
-IR. XTC cells, grown on plates to 60%
confluency, were treated with either 0 or 10 Gy of
-IR with the use
of a cesium irradiator. After a brief recovery period, the cells were
collected, lysed, and immunoprecipitated with
X53BP1 that had been
linked to protein A beads. After thoroughly washing the pellet, the
samples were split into two equal portions. One part was treated with
buffer, and the second was treated with
phosphatase. After
incubation, the samples were processed for electrophoresis on 3-8%
gradient gels. In the absence of
-IR, X53BP1 migrates at ~300 kDa
(Fig. 4, lanes 1 and 2). When exposed to 10 Gy of
-IR, X53BP1 appears as
even a slower migrating form (Fig. 4, lane 3).
This retarded form of X53BP1 can be reversed back to its basal state
through treatment with
-phosphatase (Fig. 4), indicating that the
-IR-induced modification of the protein is due to phosphorylation.
Additionally, X53BP1 is also modified via phosphorylation in the
absence of DNA damage, since it appears to have a slightly slower
mobility in gradient gels relative to the
-phosphatase-treated
sample (Fig. 4, compare lanes 1 and 2). Other factors, such as cyclin-dependent
kinases, may be responsible for this. Taken together, our results
establish X53BP1 as a new member of the
-IR DNA damage response
pathway.
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Fig. 4.
DNA damage-dependent
phosphorylation of X53BP1 in vivo. XTC cells were
treated with either 0 (lanes 1 and 2)
or 10 Gy of -IR (lanes 3 and 4).
Lysed supernatants were immunoprecipitated with
X53BP1 and split
into two equal halves. One half received buffer only (lanes
1 and 3), and the other was treated with 1.0 units of
-phosphatase (lanes 2 and
4). Immunoprecipitates were electrophoresed on 3-8%
gradient gels and immunoblotted with
X53BP1 as shown. Various
electrophoretic forms of X53BP1 are indicated by the
brackets. The position of the 209-kDa marker (myosin heavy
chain) is indicated with an arrow.
X53BP1 and a fluorescein-conjugated secondary IgG. Staining with either secondary antibody alone or with other nonspecific IgG molecules
produced no signal (not shown). This, in conjunction with the fact that
X53BP1 recognizes one major band in XTC extracts (Fig.
3A), suggests that the immunostaining data specifically represents X53BP1. The staining pattern for X53BP1 agrees with the
previously reported pattern for human 53BP1 (58). X53BP1 possesses a diffuse cytoplasmic staining pattern (Fig.
5, A and B).
Moreover, X53BP1 appears to localize to a small number of large nuclear
foci (1-3) in about 20% of the cells (Fig. 5A, see
arrows). To observe any changes in the subcellular
localization of X53BP1 that may occur in response to DNA damage, we
exposed XTC cells to 10 Gy of
-IR. After a recovery period, the
cells were processed for IF. After examining a few hundred cells, it appears that many more, smaller foci are present in nuclei that have
been exposed to the DNA-damaging agent (Fig. 5B, see
arrows), suggesting that X53BP1 is redistributed within the
nucleus in response to
-IR. These foci are absent in nonirradiated
samples. Since about 20% of the examined cells possess multiple
nuclear foci, this suggests that the focal localization of X53BP1 may occur at a discrete point(s) within the cell cycle (i.e. S
phase).
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Fig. 5.
Subcellular localization of X53BP1 during the
DNA damage response. XTC cells were processed for indirect
immunofluorescence with X53BP1 as described under "Experimental
Procedures" in the absence of DNA damage (A) or in the
presence of 10 Gy of
-IR (B). The green
area (fluorescein isothiocyanate) represents staining specifically
associated with
X53BP1, and the blue represents nuclear
DNA staining with Hoescht 33258. The scale bar
represents 25 µm.
-IR DNA damage, we asked if X53BP1
could serve as a substrate for the ATM kinase. It has been well
established that ATM is activated in response to this type of genotoxic
insult (7, 8). To show this, we generated a series of GST fusion
proteins spanning X53BP1 (Fig. 5). Such an approach has been previously
adopted to demonstrate the ATM and Chk2-dependent
phosphorylation of BRCA1 (11, 77). To perform this assay, 293T cells
were transfected with a FLAG-tagged cDNA encoding either the wild
type ATM gene (WT) or a kinase-dead (KD) version of the enzyme
(7, 8). After 48 h, the cells were harvested and lysed in TGN
buffer (8). After a preclearing step with protein G-agarose beads,
either WT or KD ATM protein was immunoisolated, extensively washed as described (8, 11), and incubated with candidate GST-X53BP1 fusion
substrate proteins in the presence of radiolabeled ATP.
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Fig. 6.
ATM-dependent phosphorylation of
X53BP1 in vitro. A, schematic
representation of GST-XBP1 fusion substrates utilized in an assay for
ATM kinase activity (7, 8). Hatched lines
represent approximate positions of (S/T)Q motifs. Five fusion proteins
as shown were created for the assay. The following aa residues are
encoded for by the GST-XBP1 fusion proteins. GST-XBP1-1, aa 1-338;
GST-XBP1-2, aa 393-821; GST-XBP1-3, 815-1206; GST-XBP4, 1201-1657;
GST-XBP5, 1652-2,055. B, affinity-purified fusion proteins
were processed for ATM kinase assays (7, 8) with either wild type ATM
(odd lanes) or a KD version (even
lanes) as shown. As a control, a GST fusion protein with
Brca1 residues 1351-1552 was used (lanes 1 and
2; Ref. 11). The arrows in lane
3 represent discrete phosphorylated species of GST-XBP1-1.
Molecular mass markers are indicated on the left in
kilodaltons. Lower molecular weight species represent degradation
products that copurify from E. coli lysates.
-IR with a cesium
irradiator, allowed to recover, and then processed for
immunoprecipitation and immunoblotting analysis with the M2/M5
FLAG-specific antibodies. If 53BP1 is phosphorylated by
-IR
activated ATM, then slower mobility forms of 53BP1 might be expected to
be seen in the form of shifts in the molecular weight of the protein.
In the absence of
-IR, pZX-12-transfected 293T cells were found to
generate multiple forms of H53BP1-524 (Fig. 7B,
lane 1), reminiscent of the behavior often
observed for X53BP1 in cell-free extracts (Fig. 3A). Because
these bands are reversible through treatment with
phosphatase
enzyme (Fig. 7B, lane 2), it appears
that H53BP1-524 is phosphorylated in its basal state by endogenous
enzymes within 293T cells. Despite this background level of
phosphorylation for FLAGBP1-524, higher molecular weight, shifted
forms of the protein are clearly apparent in
-IR-treated samples
that have been cotransfected with ATM WT but not with a plasmid
encoding the kinase-defective enzyme (Fig. 7B,
lane 7; see arrow). Such proteins
often appear as diffuse species. We confirmed that these proteins are
phosphorylated species of 53BP1, since treatment with
-phosphatase
completely reverses the induced mobility shift. Collectively, our
results suggest that 53BP1 is a targeted by ATM during the DNA damage
response.
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Fig. 7.
ATM-dependent phosphorylation of
human 53BP1 in vitro and in
vivo. A, in vitro kinase assay of
a GST fusion protein with GST and human 53BP1 aa 1-524 (HBP1-524). 1 µg of HBP1-524 was assayed as a substrate for either wild type ATM
(lane 1) or a KD version of the enzyme
(lane 2). Lower molecular weight species are the
result of degradation products commonly associated with GST fusion
proteins. B, in vivo assay for
ATM-dependent phosphorylation of pZX-12-encoded
FLAGBP1-524. 293T cells were transfected with 2.5 µg of pZX-12 alone
(left panel) or with an equal amount of ATM-WT
(middle panel) or ATM-KD (right
panel) encoding plasmids. Some samples were treated with 50 Gy of -IR (lanes 3, 4,
7, 8, 11, and 12).
FLAGBP1-524 was isolated by immunoprecipitation and evaluated for
modification by electrophoretic mobility shifts in 6% SDS gels in the
absence (odd lanes) or presence of 1.0 units of
-phosphatase (even lanes). The
arrow represents FLAGBP1-524 shifted bands.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-IR. As such, recent advancements into the mechanisms
of checkpoint control in a range of organisms from yeast to humans have
led to unexpected connections between DNA recombination/repair proteins and cell cycle control (22). The inability to repair DSBs poses a
significant threat to genomic instability. Such genetic
frailties have been observed in cells with mutations in a variety of
genes encoding proteins that participate in the biological response to
this type of DNA damage. Many of the proteins involved in the biological response to
-IR are controlled in some manner through ATM-dependent phosphorylation. ATM phosphorylates numerous
substrates, many of which participate in cell cycle control and
repairing DSBs. These include BRCA1, Cds1/Chk2, p95/Nbs1, and p53 (7, 8, 10, 11, 13, 14, 77). In this study, we have characterized 53BP1
proteins from both frog and human. Our data classify 53BP1 as a
component in the DNA damage response pathway.
-IR, an agent known to create chromosomal breaks, can induce the
phosphorylation of X53BP1 in vivo. We also provide evidence that the N-terminal region of human 53BP1 is phosphorylated in response
to
-IR in vivo. Our data suggest that at least two
kinases phosphorylate X53BP1 as the protein is phosphorylated in both the absence and the presence of
-IR DNA damage. Both the frog and
human 53BP1 orthologs contain numerous phosphorylation sites for ATM
kinase family members, particularly in their N-terminal regions (59).
This includes ATM, ATR, and DNA-PK. Our evidence implicates ATM as one
kinase that phosphorylates both the Xenopus and human 53BP1
proteins in vitro. The good in vitro specificity by ATM toward GST-XBP1-1 and GST-XBP1-4 suggests that these regions are promising targets of the kinase in vivo. Moreover, it
appears that ATM can also phosphorylate an N-terminal domain of human 53BP1 in a DNA damage-dependent manner in vivo.
Whether 53BP1 is also targeted by ATR or DNA-PK remains to be
determined. Collectively, our data establish X53BP1 as a protein that
participates in the response to
-IR DNA damage.
-IR, these foci appear to disperse and redistribute into smaller
ones throughout the nucleus. We observed that this phenomenon occurred
reproducibly in about 20% of the cells, perhaps indicating that the
-IR-dependent dispersal of X53BP1 nuclear foci may occur
at a discrete stage within the cell cycle, such as S phase. Presently,
we cannot prove that X53BP1 phosphorylation and its dispersal into
smaller nuclear foci in response to
-IR are related. Such X53BP1
foci increase in number in response to
-IR possibly as a result of
the protein being localized to sites of newly created DNA damage.
Previous studies with BRCA1 have demonstrated that the protein
disperses from nuclear foci as a function of various DNA-damaging
agents, including
-IR (15). The DNA damage-dependent
dispersal of BRCA1 foci has been shown to correlate very well with the
phosphorylation of the protein (15). Notably, the Cds1/Chk2 effector
kinase has been shown to be responsible, at least in part, for the
dispersal of BRCA1 nuclear foci in response to DNA damage (77). We
hypothesize that an analogous mechanism exists between 53BP1
phosphorylation and its subcellular localization and are currently
designing experiments to test this notion.
-IR. Whatever the case, it appears that 53BP1 is an important player
in the biological response to
-IR DNA damage and possibly other
types of genotoxic stress. The elucidation of its precise role in these
processes awaits further investigation.
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ACKNOWLEDGEMENTS |
---|
We are grateful to the many people who
contributed to and/or supported this project throughout its various
stages. In particular, we thank Steve Elledge and David Cortez for
numerous reagents and for support throughout the course of this study.
We are grateful to Thanos Halazonetis for communicating results prior
to publication. We thank Paul Mueller for use of the mitotic
catastrophe screen and Dr. David Lane for the kind gift of
Xenopus p53 antisera. We are indebted to Dr. Andrew Krieg
for the gift of Xenopus EF-1 for Northern loading
controls and to Dr. Ronald Kerman for permission to use the cesium
irradiator. We also thank Tamara Tripic and Jason Grier for their
efforts during the earlier stages of the project. We are also indebted
to Rob Kirken and Mike Blackburn for experimental advice and assistance
with the use of the fluorescence microscope, respectively.
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FOOTNOTES |
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* This work was supported in part by grants from the Ellison Medical Foundation and the Welch Foundation (to P. B. C.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF281071.
¶ Investigator of the Howard Hughes Medical Institute.
** Junior Research Scholar of the Ellison Medical Foundation. To whom correspondence should be addressed. Tel.: 713-500-6032; Fax: 713-500-0652; E-mail: Phillip.B.Carpenter@uth.tmc.edu.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M007665200
2 T. Halazonetis, personal communication.
3 J. C. Morales and P. B. Carpenter, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: AT, ataxia telangiectasia; DSB, double-stranded DNA break; aa, amino acid(s); kb, kilobase pair(s); RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; CSF, cytostatic factor; Gy, gray; PBS, phosphate-buffered saline; XTC, Xenopus tissue culture; IF, immunofluorescence; WT, wild type.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Elledge, S. J.
(1996)
Science
274,
1664-1672 |
2. | Hartwell, L. H., and Kastan, M. B. (1994) Science 266, 1821-1828[Medline] [Order article via Infotrieve] |
3. | Savitsky, K., Bar-Shira, S., Gilad, S., Rotman, G., Ziv, Y., Vanagaite, L., Tagle, D. A., Smith, S., Uzeil, T., and Sfez, S. (1995) Science 268, 1749-1753[Medline] [Order article via Infotrieve] |
4. | Painter, R. B., and Young, B. R. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7315-7317[Abstract] |
5. | Lavin, M. F., and Shiloh, Y. (1997) Annu. Rev. Immunol. 15, 177-202[CrossRef][Medline] [Order article via Infotrieve] |
6. | Futreal, P. A., Liu, Q., Shattuck-Eidens, D., Cochran, C., Harshmann, K., et al.. (1994) Science 266, 120-122[Medline] [Order article via Infotrieve] |
7. |
Banin, S.,
Shieh, S.-Y.,
Taya, Y.,
Anderson, C. W.,
Chessa, L.,
Smorodinsky, N. I.,
Prives, C.,
Reiss, Y.,
Shiloh, Y.,
and Ziv, Y.
(1998)
Science
281,
1674-1677 |
8. |
Canman, C. E.,
Lim, D.-S.,
Cimprich, K. A.,
Taya, Y.,
Tamia, K.,
Sakaguchi, K.,
Appella, E.,
Kastan, M. B.,
and Siliciano, J. D.
(1998)
Science
281,
1677-1680 |
9. |
Matsuoka, S.,
Huang, M.,
and Elledge, S. J.
(1998)
Science
282,
1893-1897 |
10. |
Bell, D. W.,
Varley, J. M.,
Szydlo, T. E.,
Kang, D. H.,
Wahrer, D. C. R.,
Shannon, K. E.,
Lubratovich, M.,
Verselis, S. J.,
Isselbacher, K. J.,
Fraumeni, J. F.,
Birch, J. M.,
Li, F. P.,
Garber, J. E.,
and Haber, D. A.
(1999)
Science
286,
2528-2531 |
11. |
Cortez, D.,
Wang, Y.,
Qin, J.,
and Elledge, S. J.
(1999)
Science
286,
1162-1166 |
12. | Lim, D.-S., Kim, S-T., Xu, B., Maser, R. S., Lin, J., Petrini, J. H. J., and Kastan, M. B. (2000) Nature 404, 613-617[CrossRef][Medline] [Order article via Infotrieve] |
13. | Wu, X., Ranganathan, V., Weisman, D. S., Heine, W. F., Ciccone, D. N., O'Neill, T. B., Crick, K. E., Pierce, K. A., Lane, W. S., Rathbun, G., Livingston, D. M., and Weaver, D. T. (2000) Nature 405, 477-481[CrossRef][Medline] [Order article via Infotrieve] |
14. | Zhao, S., Weng, Y.-C., Yuan, S.-S. F., Lin, Y.-T., Hsu, H.-C., Lin, S.-C. J., Gerbino, E., Song, M.-h, Zdzienicka, Z., Gatt, R. A., Shay, J. W., Ziv, Y., Shiolh, Y., and Lee, E. Y.-H. P. (2000) Nature 405, 473-477[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Scully, R.,
Anderson, S. F.,
Chao, D. M.,
Wei, W.,
Ye, L.,
Young, R. A.,
Livingston, D. M.,
and Parvin, J. D.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
5605-5610 |
16. |
Gowen, L. C.,
Avrutskaya, A. V.,
Latour, A. M.,
Koller, B. H.,
and Leadon, S. A.
(1998)
Science
281,
1009-1012 |
17. | Moynahan, M. E., Chiu, J. W., Koller, B. H., and Jasin, M. (1999) Mol. Cell 4, 511-518[Medline] [Order article via Infotrieve] |
18. | Xu, X., Weave, Z., Linke, S. P., Li, C., Gotay, J., Wang, X. W., Harris, C. C., Ried, T., and Deng, C. X. (1999) Mol. Cell 3, 389-395[Medline] [Order article via Infotrieve] |
19. |
Wang, Y.,
Cortez, D.,
Yazdi, P.,
Neff, N.,
Elledge, S. J.,
and Qin, J.
(2000)
Genes Dev.
14,
927-939 |
20. |
Wang, H.,
and Elledge, S. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3824-3829 |
21. | Haber, J. E. (1998) Cell 95, 583-586[Medline] [Order article via Infotrieve] |
22. | Petrini, J. H. J. (2000) Curr. Opin. Cell Biol. 12, 293-296[CrossRef][Medline] [Order article via Infotrieve] |
23. | Murakami, H., and Okayama, H. (1995) Nature 374, 817-819[CrossRef][Medline] [Order article via Infotrieve] |
24. | O'Connell, M. J., Walworth, N. C., and Carr, A. M. (2000) Trends Cell Biol. 7, 296-303[CrossRef] |
25. | Al-Khodairy, F., Foton, E., Sheldrick, K. S., Griffiths, D. J., Lehmann, A. R., and Carr, A. M. (1994) Mol. Biol. Cell 5, 147-160[Abstract] |
26. |
Brown, A. L.,
Lee, C. H.,
Schwarz, J. K.,
Mitiku, N.,
Piwnica-Worms, H.,
and Chung, J. H.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3745-3750 |
27. | Blasina, A. D., Weyer, D., Laus, M. C., Luyten, W. H., Parker, A. E., and McGowan, C. H. (1999) Curr. Biol. 9, 1-10[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Kumagai, A.,
Guo, Z.,
Emani, K. H.,
Wang, S. X.,
and Dunphy, W. G.
(1998)
J. Cell Biol.
142,
1559-1569 |
29. | Chaturverdi, P., Eng, W. K., Zhu, M. R., Mattern, R., Mishra, M. R., Hurle, M. R., Zhang, X., Annan, R. S., Lu, Q., Faucette, L. F., et al.. (1999) Oncogene 18, 4047-4054[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Kumagai, A.,
and Dunphy, W. G.
(1999)
Genes Dev.
13,
1067-1072 |
31. | Weinert, T. A., and Hartwell, L. H. (1988) Science 241, 317-322[Medline] [Order article via Infotrieve] |
32. | Paulovich, A. G., and Hartwell, L. H. (1995) Cell 82, 841-847[Medline] [Order article via Infotrieve] |
33. |
Siede, W.,
Friedberg, A. S.,
and Friedberg, E. C.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
7985-7989 |
34. |
Yang, S. S.,
Yeh, E.,
Salmon, E. D.,
and Bloom, K.
(1997)
J. Cell Biol.
136,
345-354 |
35. |
Sanchez, Y.,
Wong, C.,
Thoma, R. S.,
Richman, R.,
Wu, Z.,
Piwnica-Worms, H.,
and Elledge, S. J.
(1997)
Science
277,
1497-1501 |
36. |
Shimomura, T.,
Ando, S.,
Matsumoto, K.,
and Sugimoto, K.
(1998)
Mol. Cell. Biol.
18,
5485-5491 |
37. | Griffiths, D. J., Barbet, N. C., McCready, S., Lehmann, A. R., and Carr, A. M. (1995) EMBO J. 14, 5812-5823[Abstract] |
38. |
Caspari, T.,
Dahlen, M.,
Kanter-Smoler, G.,
Lindsay, H. D.,
Hofman, K.,
Papadimitriou, K.,
Sunnerhagen, P.,
and Carr, A. M.
(2000)
Mol. Cell. Biol.
20,
1254-1262 |
39. | Thelen, M. P., Venclovas, C., and Fidelis, K. (1999) Cell 96, 769-770[Medline] [Order article via Infotrieve] |
40. |
Bessho, T.,
and Sancar, A.
(2000)
J. Biol. Chem.
275,
7451-7454 |
41. | Halazonetis, T. D., and Shiloh, Y. (1999) Biochem. Biophys. Acta 1424, 45-55 |
42. | Kastan, M. B., Onyekwere, O., Sidransky, D., Vogelstein, B., and Craig, R. W. (1991) Cancer Res. 51, 6304-6311[Abstract] |
43. | Kastan, M. B., Zhan, Q., El-Diery, W., Carrier, F., Jacks, T., Walsh, W. V., Plunkett, B. S., Vogelstein, B., and Fornace, A. J. (1992) Cell 71, 587-597[Medline] [Order article via Infotrieve] |
44. |
Giaccia, A. J.,
and Kastan, M. B.
(1998)
Genes Dev.
12,
2973-2983 |
45. | Levine, A. J. (1997) Cell 88, 323-331[Medline] [Order article via Infotrieve] |
46. |
Chehab, N. H.,
Malikzay, A.,
Appel, M.,
and Halazonetis, T. D.
(2000)
Genes Dev.
14,
278-288 |
47. |
Shieh, S.-Y.,
Ahn, J.,
Tamia, K.,
Taya, Y.,
and Prives, C.
(2000)
Genes Dev.
14,
289-300 |
48. | Iwabuchi, K., Bartel, P. L., Li, B., Maraaccino, R., and Fields, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6098-6102[Abstract] |
49. | Callebaut, I., and Mornon, J. (1997) FEBS Lett. 400, 25-30[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Bork, P.,
Hoffman, K.,
Bucher, P.,
Neuwald, A. F.,
Altschul, S. F.,
and Koonin, E. V.
(1997)
FASEB J.
11,
68-76 |
51. |
Zhang, X.,
Morera, S.,
Bates, P. A.,
Whitehead, P. C.,
Coffer, A. I.,
Hainbucher, K.,
Nash, R.,
Sternberg, M. J. E.,
Lindahl, T.,
and Freemont, P. S.
(1998)
EMBO J.
17,
6404-6411 |
52. |
Saka, Y.,
Esashi, F.,
Matsusaka, T.,
Mochida, S.,
and Yanagida, M.
(1997)
Genes Dev.
11,
3387-3400 |
53. | Murray, A. W. (1991) Methods Cell Biol. 36, 581-605[Medline] [Order article via Infotrieve] |
54. |
Datta, B.,
Li, B.,
Choubey, D.,
Nallur, G.,
and Lengyel, P.
(1996)
J. Biol. Chem.
271,
27544-27555 |
55. | Lundgren, K., Walworth, N., Booher, R., Dembski, M., Kirschner, M., and Beach, D. (1991) Cell 64, 1111-1122[Medline] [Order article via Infotrieve] |
56. | Carpenter, P. B., Mueller, P. R., and Dunphy, W. G. (1996) Nature 379, 357-360[CrossRef][Medline] [Order article via Infotrieve] |
57. | Morgan, D. O. (1997) Annu. Rev. Cell Dev. Biol. 13, 261-291[CrossRef][Medline] [Order article via Infotrieve] |
58. |
Iwabuchi, K.,
Li, B.,
Massa, H. F.,
Trask, B. J.,
Date, T.,
and Fields, S.
(1998)
J. Biol. Chem.
273,
26061-26068 |
59. |
Kim, S.-T.,
Lim, D.-S.,
Canman, C. E.,
and Kastan, M. B.
(1999)
J. Biol. Chem.
274,
37538-37543 |
60. | Mueller, P. R., Coleman, T. R., and Dunphy, W. G. (1995) Mol. Biol. Cell 6, 119-134[Abstract] |
61. | Koonin, E. V., Altschul, S. F., and Bork, P. (1996) Nat. Genet. 13, 266-267[Medline] [Order article via Infotrieve] |
62. | Krieg, P. A., Varnum, S. M., Wormington, W. M., and Melton, D. A. (1989) Dev. Biol. 133, 93-100[Medline] [Order article via Infotrieve] |
63. | Amariglio, F., Tchang, F., Prioleau, M.-N., Soussi, T., Cibert, C., and Mechali, M. (1997) Oncogene 15, 2191-2199[CrossRef][Medline] [Order article via Infotrieve] |
64. | Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T., and Livingston, D. M. (1997) Cell 88, 265-275[Medline] [Order article via Infotrieve] |
65. | Soussi, T., de Fromentel, C. C., Mechali, M., May, P., and Kress, M. (1987) Oncogene 1, 71-78[Medline] [Order article via Infotrieve] |
66. | Cox, L. S., Midgley, C. A., and Lane, D. P. (1994) Oncogene 10, 2951-2959 |
67. | Hoever, M., Clement, J. H., Wedlich, D., Montenarh, M., and Knoechel, W. (1994) Oncogene 9, 109-120[Medline] [Order article via Infotrieve] |
68. | Tchang, F., Gusse, M., Soussi, T., and Mechali, M. (1993) Dev. Biol. 159, 163-172[CrossRef][Medline] [Order article via Infotrieve] |
69. | Wang, Y., Farmer, G., Soussi, T., and Prives, C. (1995) Oncogene 10, 779-784[Medline] [Order article via Infotrieve] |
70. | Newport, J., and Kirschner, M. (1982) Cell 30, 675-686[Medline] [Order article via Infotrieve] |
71. | Wallingford, J. B., Seufert, D. W., Virta, V. C., and Vize, P. D. (1997) Curr. Biol. 10, 747-757[CrossRef] |
72. |
Carpenter, P. B.,
and Dunphy, W. G.
(1998)
J. Biol. Chem.
273,
24891-24897 |
73. |
Humphrey, J. S.,
Salim, A.,
Erdos, M. R.,
Collins, F. S.,
Brody, L. C.,
and Klausner, R. D.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
5820-5825 |
74. | Adachi, Y., and Laemmli, U. K. (1992) J. Cell Biol. 119, 1-15[Abstract] |
75. | Coleman, T. R., Carpenter, P. B., and Dunphy, W. G. (1996) Cell 87, 53-63[Medline] [Order article via Infotrieve] |
76. | Clute, P., and Masui, Y. (1997) Dev. Biol. 185, 1-13[CrossRef][Medline] [Order article via Infotrieve] |
77. | Lee, J.-S., Collins, K. M., Brown, A. L., Lee, C.-H., and Chung, J. H. (2000) Nature 404, 201-204[CrossRef][Medline] [Order article via Infotrieve] |
78. |
Liu, Q.,
Guntuku, S.,
Cui, X. S.,
Matsuoka, S.,
Cortez, D.,
Tamai, K.,
Luo, G.,
Rivera0S, C.,
DeMayo, F.,
Bradley, A.,
Donehower, L. A.,
and Elledge, S. J.
(2000)
Genes Dev.
14,
1448-1459 |
79. | Smith, J. C., and Tata, J. R. (1991) Methods Cell Biol. 36, 636-654 |
80. | Fogarty, P., Campbell, S. D., Abu-Shumays, R. A., Phalle, B. S., Yu, K. R., Uy, G. L., Goldberg, M. L., and Sullivan, W. (1997) Curr. Biol. 7, 418-426[Medline] [Order article via Infotrieve] |
81. | Anderson, J. A., Lewellyn, A. L., and Maller, J. L. (1997) Mol. Biol. Cell 8, 1195-1206[Abstract] |