MOLECULAR AND CELLULAR BIOLOGY, May 2007, p. 3367–3377
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 27, No. 9
Function of a Conserved Checkpoint Recruitment Domain
in ATRIP Proteins?
Heather L. Ball,1Mark R. Ehrhardt,2Daniel A. Mordes,1Gloria G. Glick,1
Walter J. Chazin,2and David Cortez1*
Department of Biochemistry1and Departments of Biochemistry and Chemistry and Center for Structural Biology,2
Vanderbilt University, Nashville, Tennessee 37232
Received 29 November 2006/Returned for modification 29 December 2006/Accepted 16 February 2007
The ATR (ATM and Rad3-related) kinase is essential to maintain genomic integrity. ATR is recruited to
DNA lesions in part through its association with ATR-interacting protein (ATRIP), which in turn interacts with
the single-stranded DNA binding protein RPA (replication protein A). In this study, a conserved checkpoint
protein recruitment domain (CRD) in ATRIP orthologs was identified by biochemical mapping of the RPA
binding site in combination with nuclear magnetic resonance, mutagenesis, and computational modeling.
Mutations in the CRD of the Saccharomyces cerevisiae ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction,
prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA-damaging agents, and partially
compromise checkpoint signaling. These data demonstrate that the CRD is critical for localization and optimal
DNA damage responses. However, the stimulation of ATR kinase activity by binding of topoisomerase binding
protein 1 (TopBP1) to ATRIP-ATR can occur independently of the interaction of ATRIP with RPA. Our results
support the idea of a multistep model for ATR activation that requires separable localization and activation
functions of ATRIP.
ATR (ATM and Rad3-related) kinase is a protein kinase
that coordinates cellular responses to genotoxic stress. ATR
activation occurs primarily in S phase due to replication stress
induced by DNA-damaging agents or replication inhibitors.
More specifically, ATR activation is stimulated when the rep-
lication machinery encounters a DNA lesion and becomes
uncoupled (the helicase continues to unwind DNA while the
polymerase becomes stalled at the site of DNA damage) (9).
The critical factor that promotes ATR activation is believed
to be the accumulation of RPA (replication protein A)-coated
single-stranded DNA (ssDNA) (11, 33, 43). At least two sep-
arate checkpoint complexes accumulate in distinct foci that
colocalize with RPA. Rad17, a PCNA-like clamp loader pro-
tein, is recruited to RPA-ssDNA and loads the Rad9-Rad1-
Hus1 checkpoint clamp at the junction of double-stranded and
single-stranded DNA (4, 14, 53). Independently, ATR is re-
cruited by ATR-interacting protein (ATRIP), which binds the
RPA-ssDNA that accumulates at DNA lesions (3, 15, 37, 52).
ATRIP is required for ATR function, and mutation of either
ATR or ATRIP causes the same phenotypes (3, 12). The strict
requirement for ATRIP is conserved in Schizosaccharomyces
pombe (Rad3 and Rad26), Saccharomyces cerevisiae (Mec1 and
Ddc2/Lcd1/Pie1), and Xenopus laevis (xATR and xATRIP)
(13, 38, 41, 51). An N-terminal domain of ATRIP binds RPA-
ssDNA and is necessary for stable ATR-ATRIP localization to
damage-induced nuclear foci (3, 25).
The ATR signaling pathway is currently viewed as an im-
portant target for the development of cancer therapies (10, 22,
24, 32, 34). However, the mechanism by which ATR is acti-
vated remains unclear. Localization to sites of DNA damage or
replication stress has been suggested to be essential and per-
haps sufficient to promote ATR signaling. However, mutations
in ATRIP that disrupt the stable RPA-ATRIP interaction and
impair the accumulation of ATR-ATRIP complexes in DNA-
damage-induced foci have minimal effects on ATR activation
and signaling (3, 25). Furthermore, topoisomerase binding
protein 1 (TopBP1) was recently discovered to stimulate ATR
kinase activity, suggesting regulation by a means other than
localization (28). To clarify the functions of ATRIP, RPA, and
TopBP1 in mediating ATR-dependent checkpoint response we
have performed a series of biochemical and genetic experi-
ments in human and yeast systems. We report structural and
functional data that support a model for ATR activation in
which two separable ATRIP activities—localization and acti-
vation—cooperate to promote ATR signaling.
MATERIALS AND METHODS
Yeast strains. All strains used in this study are described in Table 1. Myc-Ddc2
and Myc-Ddc2 mutant strains were generated by expressing mutants from a
centromeric plasmid under the control of the endogenous DDC2 promoter in
strain DMP2995/1B (MATa sml1D::KanMX4 ddc2D::KanMX4) (38). GFP-
Ddc2?N was generated in JK8-1 (36) by use of the delitto perfetto system (47).
Strain yHB244 was generated by expressing RNR3 by use of pBAD79 and
deleting DDC2 by use of pGEM499 in the JKM179 strain (30). Myc-DDC2 and
Myc-Ddc2?N were expressed in strain yHB244 from the pNML1 centromeric
RPA-ssDNA and RPA binding. The 14 kDa and 70 kDa RPA subunits were
tagged with an His6epitope tag (45). RPA was purified from Escherichia coli by
use of nickel affinity chromatography followed by Superdex fractionation. A
20-pmol volume of biotin-labeled 69-bp single-stranded oligonucleotide was
bound to streptavidin beads and incubated with binding buffer (10 mM Tris [pH
7.5], 100 mM NaCl, 10% glycerol, 0.02% Igepal CA-630, 10 ?g/ml bovine serum
albumin) alone or with a 4 M excess of RPA in binding buffer. The RPA-ssDNA-
streptavidin beads were washed three times with binding buffer prior to use.
Hemagglutinin-ATRIP (HA-ATRIP) fragments were generated using in vitro
* Corresponding author. Mailing address: Department of Biochem-
istry, Vanderbilt University, 613 Light Hall, 23rd @ Pierce Ave., Nash-
ville, TN 37232. Phone: (615) 322-8547. Fax: (615) 343-0704. E-mail:
?Published ahead of print on 5 March 2007.
transcription/translation (Promega) and added to recombinant His-RPA or His-
RPA-ssDNA beads in binding buffer, and RPA was isolated using His-Select
(Sigma Aldrich) or ssDNA-Sepharose beads. Proteins bound to beads were
washed with binding buffer three times, eluted, and separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to blotting.
Kinase assays. ATR kinase assays were performed essentially as described
previously (28) with the following alterations. HA-ATRIP and Flag-ATR ex-
pression vectors were transfected into 293T cells and the ATR-ATRIP com-
plexes purified by immunoprecipitation. Kinase reactions were performed with
the antibody-linked ATR-ATRIP complex. Recombinant TopBP1 and RPA
heterotrimer was purified from E. coli. The Phas1 substrate was purchased from
A.G. Scientific. The in situ Rad53 autophosphorylation assay following denatur-
ation/renaturation was performed as previously described (39).
Cell culture. All cells were grown in Dulbecco modified Eagle medium
(DMEM)–7.5% fetal bovine serum. Plasmid transfections were performed with
Lipofectamine 2000 (Invitrogen). The ATRIP wild-type (WT) and ATRIP?N
293T cells were generated by retroviral infection and selection (3). ATRIP small
interfering RNA (siRNA) transfections and immunofluorescence methods were
also performed as described previously (3).
Protein purification and antibodies. Tandem affinity purification (TAP)-
Rfa1 was purified from soluble yeast extracts as described previously (1, 17).
RPA70N was kindly provided by Cheryl Arrowsmith in a pET15b vector
(Novagen) and was grown on defined M9 medium supplemented with15N-
NH4Cl and D-glucose and purified over nickel-nitrilotriacetic acid. ?H2AX
(Upstate), Myc9E10 (Covance), and HA.11 (Covance) were purchased from
the indicated companies. Rad53 antibody was a gift from Stephen Elledge.
Rfa1 antibody was a gift from Steven Brill.
NMR analysis and homology modeling. Nuclear magnetic resonance (NMR)
spectra were collected on ?100 ?M15N-RPA70N in a buffer containing 2 mM
?-mercaptoethanol, 50 mM NaCl, and 20 mM Tris–d11at pH 7.4. ATRIP1-107
and ATRIP54-70 were added at a four- to sixfold molar excess to maximize the
bound-state population of the observed component, RPA70N. NMR experi-
ments were performed at 25°C using Bruker AVANCE 500 MHz or 600 MHz
NMR spectrometers equipped with a 5 mm single axis z-gradient Cryoprobe.
Two-dimensional, gradient-enhanced15N-1H heteronuclear single-quantum cor-
relation (HSQC) spectra were recorded with 1,024 complex datum points in the
1H dimension and 96 complex points in15N dimension.1H and15N backbone
NMR assignments for RPA70N were kindly provided by Cheryl Arrowsmith.
Chromatin immunoprecipitation (ChIP) analysis. HO expression in strains
yHB245 (vector), yHB246 (Myc-DDC2), and yHB247 (Myc-Ddc2?N) containing
galactose-inducible expression of HO endonuclease was performed as described
previously (30). Cells were cross-linked with 1% formaldehyde, lysed, and son-
icated to generate DNAs with an average size of 500 bp. Myc-Ddc2 protein-DNA
complexes were isolated using Myc9E10 antibody and protein G-Sepharose
beads, washed extensively, and eluted from beads. Cross-links were reversed by
overnight incubation at 65°C. DNA was precipitated and amplified using the
following primers specific to a region adjacent to the HO break site (HO-A or
HO-B) or to a region of SMC2: HO-A1 (5?-CTCATCTGTGATTTGTGG-3?),
HO-A2 (5?-AGAGGGTCACAGCACTAATACA-3?), HO-B1 (5?-CCAGAT
TTGTATTAGACGAGGGACGGAGTGA-3?), HO-B2 (5?-AGAGGGTCA
CAGCACTAAATACAGCTCGAAT-3?), SMC2-1 (5?-AAGAGAAACTTTA
GTCAAAACATGGG-3?), and SMC2-2 (5?-CCATCACATTATACTAACT
Characterization of ATRIP and RPA binding domains. Our
analysis started with an examination of the binding of ATRIP
fragments to RPA in the absence and presence of ssDNA.
Previous analysis of ATRIP identified at least three domains:
an N-terminal RPA-ssDNA binding domain, a dimerization
domain predicted to fold into a coiled-coil structure, and a
C-terminal ATR-interaction domain (2, 3). HA-tagged, intact
ATRIP and ATRIP fragments spanning the various domains
were generated using a coupled transcription/translation sys-
tem. These ATRIP proteins were added to purified His-tagged
RPA heterotrimer bound to ssDNA displayed on Sepharose
beads or His-tagged RPA heterotrimer bound to nickel beads.
After incubation and washing, the bound ATRIP proteins were
detected by Western blot analysis. We found that all ATRIP
fragments containing the N-terminal 107 amino acids bound
well to RPA-ssDNA and His-RPA in the absence of DNA
(Fig. 1A and B). Thus, the first 107 amino acids of ATRIP
contain a protein-protein interaction domain that mediates
binding to the RPA heterotrimer.
HA-ATRIP fragments lacking this N-terminal RPA binding
domain of ATRIP (N-RBD) were deficient in binding RPA-
ssDNA and His-RPA (Fig. 1A and B). Long exposures of
Western blots did show a small degree of association of these
ATRIP fragments with RPA. In fact, all of the protein frag-
ments that we tested, including a fragment of Brca1, bound
weakly to RPA-ssDNA (Fig. 1A). These interactions may re-
flect additional RPA-ssDNA binding domains on ATRIP, as
has been previously reported (37).
To determine which subunit of the RPA heterotrimer inter-
TABLE 1. Yeast strains used in this study
Strain Description-genotypeReference or source
MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3 sml1::KanMX4
DMP2995/1B ?p1220: Myc-URA3-CEN?
DMP2995/1B ?pNML1: Myc-DDC2-URA3-CEN?
DMP2995/1B ?pHB126: Myc-ddc2?N-URA3-CEN?
DMP2995/1B ?pHB157: Myc-ddc2D12KD13K-URA3-CEN?
DMP2995/1B ?pHB155: Myc-ddc2NAAIRS14-19-URA3-CEN?
MATa his3-1 leu2-0 met15-0 ura3-0 Rfa1-TAP
Rfa1-TAP ?pNML1: Myc-DDC2-URA-CEN?
Rfa1-TAP ?pHB126: Myc-ddc2?N-URA-CEN?
Rfa1-TAP ?pHB157: Myc-ddc2D12KD13K-URA3-CEN?
Rfa1-TAP ?pHB155: Myc-ddc2NAAIRS14-19-URA3-CEN?
ho? MATa hml?::ADE1 hmr?::ADE1 ade1-100 leu2-3,112 lys5 trp1::hisG?
JKM179 ddc2?::LEU2 ?pBAD79: RNR3-TRP1-CEN?
yHB244 (GAL-HO, ?ddc2) ?p1220: Myc-URA3-CEN?
yHB244 (GAL-HO, ?ddc2) ?pNML1: Myc-DDC2-URA3-CEN?
yHB244 (GAL-HO, ?ddc2) ?pHB126: Myc-ddc2?N-URA3-CEN?
mat? can1 ade2 trp1 his3 ura3 leu2 lys5 cyh2 ade3::GalHO adh4::HO site::HIS3
yJK8-1 ddc2?N-GFP ?pBAD79: RNR3-TRP1-CEN?
3368 BALL ET AL.MOL. CELL. BIOL.
acts with ATRIP, we purified recombinant RPA domains in-
dividually or in combination as His-tagged proteins (Fig. 1C).
Using pull-down assays with in vitro-translated ATRIP pro-
teins, we found that full-length HA-ATRIP or the isolated
N-RBD bound only to RPA fragments containing the N-ter-
minal RPA70 oligonucleotide/oligosaccharide (OB) fold do-
main (RPA70N) (Fig. 1D). No significant binding to other
RPA domains was detectable, and no binding of ATRIP?N
(ATRIP108-791) protein lacking the N-RBD to any RPA frag-
ment (Fig. 1D) was detectable in this assay. Taken together,
these data suggest that the ATRIP N-RBD interacts directly
with the 70N domain of RPA.
A conserved acidic domain in the ATRIP N terminus inter-
acts with the basic cleft of the RPA70N OB fold. The specific
residues involved in the interaction of RPA and ATRIP were
identified using an NMR chemical-shift-mapping approach.
This strategy involves monitoring NMR chemical shifts of one
protein over the course of a titration with a binding partner.
Measurement of the RPA15N-1H-HSQC NMR spectrum of
15N-enriched RPA70N as ATRIP N-RBD after titration in
solution showed that only a subset of the RPA70N signals was
affected (Fig. 2A). The observation of effects in the fast-to-
intermediate-exchange regimen on the NMR timescale sug-
gests that binding was occurring with a dissociation constant in
the low micromolar range. When the chemical shifts are
mapped onto the crystal structure of RPA70N (7), it is appar-
ent that ATRIP N-RBD interacts within the basic cleft of
RPA70N (Fig. 2B).
Initial insight into characteristics of the RPA70N binding
site of the ATRIP N-RBD was obtained from sequence anal-
ysis. When the N termini of five ATRIP orthologs were
aligned, minimal sequence similarity was observed, with the
notable exception of a small, acidic region spanning approxi-
mately 15 amino acids (Fig. 2C). On the basis of putative
electrostatic complementarity, we hypothesized that this small
acidic region made contact with the basic surface in the cleft of
the OB fold of RPA70N. To test this hypothesis, an ATRIP
peptide spanning the conserved acidic region (ATRIP54-70)
was synthesized, and the RPA70N titration was repeated. The
titration with the peptide perturbed most of the same residues
FIG. 1. ATRIP N terminus binds RPA70N in vitro. HA-tagged, full-length ATRIP or ATRIP fragments generated using a coupled transcrip-
tion/translation system were incubated with single-stranded DNA bound to Sepharose beads in the presence (?) or absence (?) of purified RPA
(A) or His-RPA bound to nickel beads (B). After washing, the bound proteins were eluted, separated by SDS-PAGE, blotted, and probed with
HA antibody. Input (In) (10%) data are included for comparison. (C and D) Purified, recombinant His-tagged RPA domains were added to in
vitro translation reaction mixtures containing HA-ATRIP, HA-ATRIP?N (C), or HA-ATRIP1-107 (D). Protein complexes were isolated using
nickel beads, separated by SDS-PAGE, blotted, and probed using an HA antibody. (E) Diagram of RPA heterotrimer. Black bars above protein
segments indicate protein interaction domains.
VOL. 27, 2007 FUNCTIONS OF RPA AND TOPBP1 IN ATR-ATRIP SIGNALING 3369
as the titration with ATRIP N-RBD, indicating that the
ATRIP peptide binds in the same manner within the basic cleft
of RPA70N (Fig. 2D). In addition, we analyzed the binding of
ATRIP1-107 containing charge-reversal mutations at positions
D58 and D59 to RPA70N by use of NMR. This mutant binds
much more weakly than wild-type ATRIP.
The basic cleft of RPA70N has been shown to bind peptides
that can mimic DNA in a manner similar to the binding of
ssDNA to the A and B domains of RPA70 (5, 7). RPA70N
binds an acidic helical peptide of p53, and the crystal structure
of the p53 peptide bound in the cleft was determined by a
method previously reported (7). Alignment of this p53 peptide
with ATRIP54-70 indicates significant homology between the
two peptides (Fig. 2E). Therefore, the crystal structure of
RPA70N bound to the p53 peptide was used to generate a
homology model for the ATRIP peptide-RPA70N interaction.
The strategy involved using the backbone coordinates of the
RPA70N and the p53 peptide along with the side chains of
FIG. 2. A conserved acidic domain in the ATRIP N terminus interacts with the basic cleft of RPA70N. (A) The15N-1H-HSQC NMR spectrum
of15N-enriched-RPA70N in the absence (blue) and presence (red) of ATRIP1-107. (B) RPA70N residues perturbed (blue) upon addition of
ATRIP1-107 mapped onto the crystal structure of RPA70N (PDB accession 2B3G). (C) Sequence alignment of the conserved acidic region in the
N terminus of five ATRIP orthologues. (D) The15N-1H-HSQC NMR spectrum of15N-enriched RPA70N acquired in the absence (blue) and
presence (red) of ATRIP54-70. (E) Alignment of the p53 and ATRIP peptides used in homology modeling. (F) Space-filling diagram of RPA70
and ATRIP55-66 (red), with the residues of RPA70N in the ATRIP binding pocket colored blue. (G) Predicted electrostatic interactions between
basic RPA70N basic residues K88 and R41 with ATRIP acidic residues D58 and D59.
3370 BALL ET AL.MOL. CELL. BIOL.
RPA70N from the crystal structure. The p53 amino acid side
chains were replaced with the ATRIP amino acid 55 to 66 side
chains, and the best fit of the ATRIP peptide into the con-
strained RPA70N was determined using ROSETTA (Fig. 2F)
The model predicts that there are several specific electro-
static interactions between the acidic residues on ATRIP and
the basic residues on RPA. In particular, the absolutely con-
served aspartic acid residues D58 and D59 of ATRIP are likely
to make contact with R41 and K88 of RPA70N (Fig. 2G). Basic
residues at these positions in RPA are highly conserved. The
NMR data and molecular modeling are fully consistent with
the previously described pull-down experiment results, indicat-
ing that the N terminus of ATRIP binds directly to RPA70N.
Importantly, these data create a structural framework within
which specific ATRIP-RPA binding mutants can be designed
and used for functional analysis.
The N-RBD of human ATRIP is conserved in the S. cerevi-
siae ATRIP ortholog Ddc2. The functional consequences of
disrupting the ATRIP-RPA interaction in human cells were
previously characterized using an ATRIP mutant lacking
the entire N-RBD (ATRIP?N). Unlike wild-type ATRIP,
ATRIP?N has a severe defect in localizing to damage- or
replication stress-induced nuclear foci (3). Despite this local-
ization defect, cells depleted of endogenous ATRIP and com-
plemented with ATRIP?N exhibit normal ATR-dependent
signaling following DNA damage (3). The only checkpoint
defect that we have uncovered in the ATRIP?N-expressing
cells is a slight delay in recovery from hydroxyurea (HU)-
induced stalling of replication (H. L. Ball, unpublished data).
The use of RNA interference in endogenous ATRIP synthesis
is not 100% effective, and the results are variable from cell to
cell. In addition, the level of retrovirally expressed ATRIP or
ATRIP?N after integration of the retroviral vector is variable
and not equivalent to the endogenous protein levels (3). These
technical limitations to performing genetic analysis of human
cell cultures may confound our ability to detect the phenotypic
consequences of abrogating the ATRIP-RPA interaction.
Therefore, we sought to examine the physiological role of the
ATRIP-RPA interaction in the genetic system of another or-
The RPA binding domain of ATRIP is N terminal to the
predicted coiled-coil domain. To determine whether the equiv-
alent region of yeast ATRIP (Ddc2) mediates binding to yeast
RPA70 (Rfa1), we deleted 42 amino acids N terminal to the
predicted coiled-coil domain of Ddc2 (Ddc2?N) (Fig. 3A).
Binding of Ddc2 and Ddc2?N to Rfa1 was assayed using co-
immunoprecipitation. Myc-tagged Ddc2 (WT) or Ddc2?N
(?N) was expressed from plasmids under the control of the
endogenous DDC2 promoter in ?ddc2 cells containing HA-
tagged Mec1. Myc-Ddc2 and Myc-Ddc2?N were immunopre-
cipitated using a Myc antibody, and the coassociated Rfa1 and
Mec1 were assayed by Western blotting. As expected, both
Mec1 and Rfa1 were coimmunoprecipitated with Ddc2 (Fig.
3B). In comparison, Rfa1 association with Ddc2?N was greatly
reduced, although Ddc2?N continued to bind Mec1 (Fig. 3B).
These data suggest that the N terminus of Ddc2 is required for
a stable Ddc2-Rfa1 interaction. The amount of Rfa1-associ-
ated Ddc2 was not altered by exposing cells to UV damage,
suggesting that the Ddc2-Rfa1 interaction may not be regu-
lated by DNA damage (Fig. 3B). However, these experiments
utilized soluble extracts, so it is possible that the interaction
with DNA-bound RPA is regulated.
Sequence alignment of human and yeast ATRIP indicates
that the small acidic region in the ATRIP N-RBD is conserved
(Fig. 2C). The homology model generated from NMR data
predicts that the absolutely conserved aspartic acid residues in
this region (D12 and D13 in Ddc2) could make contacts with
conserved basic amino acids on yeast Rfa1. Therefore, we
hypothesized that mutating these residues would disrupt Ddc2-
Rfa1 binding. To test this hypothesis a Ddc2 mutant was gen-
erated with aspartic acid-to-lysine charge-reversal mutations in
these two aspartic acids (Ddc2DK) (Fig. 3C). In addition, a
mutant (Ddc2N14) was generated replacing Ddc2 residues 14
to 19 with a peptide (NAAIRS) that is known to adopt a
helical conformation (Fig. 3C). Myc-Ddc2, Myc-Ddc2?N, Myc-
Ddc2DK, or Myc-Ddc2N14 was expressed in a yeast strain
containing TAP-tagged Rfa1. TAP purification of Rfa1 protein
complexes indicated they contained Myc-Ddc2 whether or not
cells were pretreated with methyl methanesulfonate (MMS)
FIG. 3. Ddc2-Rfa1 interaction requires the conserved acidic region
of Ddc2. (A) Schematic diagram of ATRIP, RPA-binding mutant
ATRIP (ATRIP?N), Ddc2, and Ddc2?N. Locations of predicted
coiled-coil domains (gray) are indicated. (B) ?ddc2 HA-MEC1 yeast
containing myc-vector (Vec), myc-Ddc2 (WT), or myc-Ddc2?N (?N)
were exposed to 0 (?) or 60 (?) J/m2UV and harvested 1 h later. Myc
immunoprecipitates from soluble extracts were separated by SDS-
PAGE, blotted, and probed with Myc, Rfa1, and HA antibodies.
(C) Alignment of conserved acidic region in the N terminus of ATRIP
and Ddc2 and schematic of mutations generated in this region of Ddc2.
(D) Yeasts containing TAP-Rfa1 and Ddc2 (WT), Ddc2?N (?N),
Ddc2DK (DK), or Ddc2N14 (N14) were damaged with 0.01% MMS
(?) or left untreated (?) and harvested 1 h later. Cells were lysed, and
TAP-Rfa1 was isolated using immunoglobulin G beads. TAP protein
complexes were separated by SDS-PAGE and Western blotted using
Myc (Ddc2) and Rfa1 antibodies.
VOL. 27, 2007 FUNCTIONS OF RPA AND TOPBP1 IN ATR-ATRIP SIGNALING3371
(Fig. 3D). In contrast, TAP-Rfa1 purifications contained min-
imal Myc-Ddc2?N, Myc-Ddc2DK, or Myc-Ddc2N14 protein
(Fig. 3C). These results confirm that the conserved acidic re-
gion in the N terminus of Ddc2 is required for a stable Ddc2-
Ddc2-Rfa1 interaction is required for localization of Ddc2 to
sites of DNA damage. Human ATRIP lacking the N-terminal
RPA binding domain (ATRIP?N) is defective in DNA-dam-
age-induced focus formation (3). To determine whether the
interaction between the N terminus of Ddc2 and Rfa1 is also
required for the localization of Ddc2 to sites of DNA damage
we assayed Ddc2 localization by use of ChIP analysis and focus
formation. To do this we used the inducible HO nuclease
system, which introduces a single double-strand break in the
yeast genome (29). The induction of a double-strand break in
?ddc2, DDC2, or ddc2?N yeast strains harboring a galactose-
inducible HO endonuclease was diagnosed by comparing PCR
products generated using primer sets adjacent to or spanning
the HO cleavage site, and the results were equal in all strains.
One hour after HO induction, cells were treated with cross-
linking agent, lysed, and sonicated, and Myc-Ddc2 protein-
DNA complexes were isolated by immunoprecipitation. Myc-
Ddc2-bound DNA fragments were recovered and amplified by
PCR using two different HO primer sets (HO-A and HO-B)
adjacent to the HO cleavage site. As a control we amplified a
region of the SMC2 gene that is on a chromosome different
than that with the HO cleavage site. WT Ddc2 specifically
accumulated at the HO cleavage site but not at the SMC2 site
after induction of the HO endonuclease (Fig. 4A). Compared
to wild-type Ddc2 results, the accumulation of Ddc2?N at the
HO break site was severely reduced although not completely
abrogated (Fig. 4A). Quantitation of the results of ChIP
experiments indicated that Ddc2 binding to the HO cleavage
site is fivefold greater than Ddc2?N binding. Ddc2 and
Ddc2?N were expressed at equal levels, and the levels of
efficiency of immunoprecipitation were equal in all samples
To determine whether the defect in Ddc2?N accumulation
at sites of DNA double-strand breaks as detected using ChIP
correlated with a defect in accumulation of Ddc2?N into
DNA-damage-induced nuclear foci we fused a C-terminal
green fluorescent protein (GFP) tag onto Ddc2 and Ddc2?N.
Equal expression of GFP-Ddc2 and GFP-Ddc2?N was as-
sessed by Western blotting with an antibody specific to the
GFP tag (Fig. 4C). HO-endonuclease expression in GFP-
DDC2 and GFP-ddc2?N strains was induced and Ddc2 local-
ization monitored by fluorescence microscopy. Induction of a
DNA break caused GFP-Ddc2 to accumulate into one distinct
FIG. 4. Ddc2 lacking the N-terminal Rfa1 binding domain is defective in localizing to sites of DNA damage. (A) ?ddc2 yeasts transformed with
a centromeric plasmid expressing Myc-Ddc2 or Myc-Ddc2?N from the DDC2 promoter and harboring a galactose-inducible HO endonuclease
were grown to log phase in raffinose-containing media. Galactose (GAL) or glucose (GLU) was added to induce or suppress HO-endonuclease
expression. One hour after sugar addition, cells were cross-linked using formaldehyde and harvested. Cells were lysed and sonicated, and
Myc-Ddc2 proteins were immunoprecipitated with a myc antibody. Cross-links were reversed, and associated DNA sequences were amplified by
PCR using primers specific to regions adjacent to the HO break site (HO-A, HO-B) or to the SMC2 gene (SMC2). Samples were prepared in
duplicate. Input samples represent 5% of input into immunoprecipitation reactions. (B) Equal volumes of immunoprecipitation reaction mixtures
before (pre) or after (post) isolation of Myc-Ddc2 proteins were separated by SDS-PAGE, blotted, and probed with a myc antibody. (C) Extracts
from ?ddc2 yeast, or from yeast expressing GFP-Ddc2 (WT) or GFP-Ddc2?N (?N), were separated by SDS-PAGE and Western blotted using
a GFP antibody. (D) Yeasts expressing GFP-Ddc2 (WT) or GFP-Ddc2?N (?N) and galactose-inducible HO endonuclease were grown to log
phase in liquid culture. Glucose (GLU) or galactose (GAL) was added to suppress or induce DNA double-strand-break formation. GFP
fluorescence was visualized on a Zeiss Axioplan fluorescent microscope. (E) Quantitation of HO-induced focus formation of GFP-Ddc2 or
GFP-Ddc2?N 4 h or 6 h after induction of HO endonuclease expression. Error bars represent standard deviations of the results from three
3372 BALL ET AL.MOL. CELL. BIOL.
focus per cell in 40% and 42% of the cells at 4 h and 6 h after
HO induction, respectively (Fig. 4D and E). Unlike GFP-
Ddc2, GFP-Ddc2?N formed a focus in only 13% and 19% of
cells after 4 h and 6 h of HO induction, respectively (Fig. 4D
and E). Additionally, in cells that did demonstrate HO-in-
duced GFP-Ddc2?N foci, the foci were noticeably smaller than
GFP-Ddc2 foci (Fig. 4D). Taken together, these data demon-
strate that, consistent with the role of ATRIP-RPA interaction
in human cells, Ddc2-Rfa1 interaction is required for efficient
localization of Ddc2 to sites of DNA damage. Since the N-
RBD of both ATRIP and Ddc2 is required for recruitment of
the ATR-ATRIP/Mec1-Ddc2 checkpoint complexes to DNA
lesions, we have named this domain the checkpoint protein
recruitment domain (CRD).
Disruption of the Ddc2-Rfa1 interaction sensitizes cells to
DNA damage. To examine the function of the Ddc2-Rfa1 inter-
action in Mec1-dependent checkpoint signaling, we first deter-
mined whether disrupting binding sensitized cells to replication
stress or DNA damage. ?ddc2 yeasts expressing Ddc2, Ddc2?N,
Ddc2DK, or Ddc2N14 were grown to log phase in liquid culture
and plated onto media containing increasing amounts of HU or
MMS. Yeasts lacking Ddc2 altogether are extremely sensitive to
even low doses of HU or MMS (Fig. 5A and B). In contrast, none
of the mutant ddc2 strains were sensitive to low doses of HU, and
only a very small difference was visible compared to the DDC2
strain results at the highest HU concentration (Fig. 5A). The
difference in sensitivity to genotoxic agents between WT and
mutant strains was more apparent in response to the MMS.
ddc2?N, ddc2DK, and ddc2N14 strains were more sensitive to
high doses of MMS than the DDC2 strain but much less sensitive
cell viability was reduced to 3% compared to 23% for DDC2 and
less than 0.01% for ?ddc2 yeast (Fig. 5B). At 0.15% MMS there
was a difference of an order of magnitude in the viability of
ddc2?N ddc2DK and ddc2N14 strains compared to WT DDC2
results (Fig. 5B). These results suggest that the Ddc2 CRD is
FIG. 5. Disruption of Ddc2-Rfa1 interaction impairs the DNA damage response. (A and B) Yeasts lacking Ddc2 (Vector) or expressing Ddc2
(WT), Ddc2?N (?N), Ddc2DK (DK), or Ddc2N14 (N14) were grown to log phase in liquid culture and plated onto rich media containing
increasing amounts of HU (A) or MMS (B). Percent viability was calculated as the number of colonies surviving at each dose compared to the
number of colonies that survived on plates lacking HU or MMS. Data represent the averages of the results of three experiments. Standard
deviations were smaller than symbol width in most cases. (C to E) Yeast strains were arrested in G1with alpha factor and released into rich media
in the presence of 200 mM HU (C and E) or in the indicated concentration of HU (D). Cells were harvested 1 h (C and D) or at the indicated
times (E) after G1release, and trichloroacetic acid was precipitated. Lysates were separated by SDS-PAGE, blotted, and probed with Rad53 or
Myc antibodies. (F and G) ?ddc2 (V), DDC2 (WT), or ddc2?N (?N) yeasts were grown to log phase in liquid culture, arrested in G1with alpha
factor, and released into media containing the indicated doses of MMS and harvested 1 h post G1release (F) or at the indicated various time points
after G1release (G). Cells were lysed, and proteins were separated by SDS-PAGE, blotted, and probed with Rad53 antibody. (G) Membranes
containing immobilized proteins were subjected to in situ autophosphorylation to assay Rad53 autophosphorylation activity.
VOL. 27, 2007 FUNCTIONS OF RPA AND TOPBP1 IN ATR-ATRIP SIGNALING3373
important for survival of cells following exposure to the DNA-
alkylating agent MMS.
To directly examine the role of the Ddc2 CRD in checkpoint
signaling, we tested the ability of wild-type Ddc2 or Ddc2
mutants to support Mec1-dependent Rad53 phosphorylation.
Yeast were grown to log phase, arrested in G1with alpha
factor, released in the presence or absence of 200 mM HU, and
harvested at various time points after release. Cell lysates were
generated, and proteins were separated by SDS-PAGE and
blotted using antibodies to Rad53. Rad53 phosphorylation is
detectable by an electrophoretic mobility shift and is defective
in ?ddc2 yeast, as seen by the absence of a slower-migrating
form of Rad53 (Fig. 5C). Consistent with the lack of HU
sensitivity, Ddc2-Rfa binding mutants Ddc2?N, Ddc2DK, and
Ddc2N14 all support Rad53 phosphorylation after exposure to
HU as efficiently as Ddc2 (Fig. 5C). Detailed time course and
dose-response experiments also failed to detect a significant
Rad53 activation defect in the Rfa1-binding mutant strains in
response to HU (Fig. 5D and E). These results are consistent
with the effects of equivalent mutations in human ATRIP
which fail to disrupt ATR signaling in response to HU (3).
In contrast, we did observe an attenuation of Mec1 signaling
in these yeast strains in response to the presence of MMS.
Strains were grown to log phase, arrested in G1with alpha
FIG. 6. TopBP1 activates ATR-ATRIP complexes independently of RPA. (A) Wild-type ATR-ATRIP or ATR-ATRIP?N complexes were
isolated from transfected 293T cells and incubated with recombinant wild-type TopBP1 978–1286 (WT) or TopBP1 978–1286 W1145R (WR),
Phas1 substrate, and [?-32P]ATP. Kinase reaction mixtures were separated by SDS-PAGE, stained with Coomassie blue, and exposed to film (32P).
A duplicate gel was blotted and probed with anti-ATRIP and anti-ATR antibodies (WB). (B) Wild-type ATR-ATRIP or kinase-dead ATR-ATRIP
immune complexes were isolated from transfected 293T cells and incubated with recombinant TopBP1 and/or RPA heterotrimer in the presence
of Phas1 substrate and [?-32P]ATP. Kinase reaction mixtures were separated by SDS-PAGE, stained with Coomassie blue or blotted, and exposed
to film (32P) or probed with anti-ATR antibodies (WB). (C and D) 293T cells stably expressing siRNA-resistant ATRIP, ATRIP?N, or empty
vector control were transfected with ATRIP siRNA to deplete endogenous ATRIP. Two days after siRNA transfection, the cells were transfected
with GFP-TopB1 978–1286 expression construct. Twenty-four hours later the cells were fixed and stained with antibodies to ?H2AX. (C) Rep-
resentative images collected on a Zeiss Axioplan microscope with the same exposure times. (D) Quantitation of the percentages of the
GFP-TopBP1-expressing cells that contained phosphorylated H2AX. Error bars represent standard deviations. The inset presents a Western blot
showing the relative expression levels of ATRIP and ATRIP?N.
3374BALL ET AL.MOL. CELL. BIOL.
factor, and released into media containing various doses of
MMS. Phosphorylated Rad53 is visible in DDC2 cells after the
addition of 0.01% MMS (Fig. 5F). However, Rad53 phosphor-
ylation is attenuated in ddc2?N cells, indicating that optimal
Rad53 phosphorylation after exposure to MMS depends upon
Ddc2-Rfa1 binding (Fig. 5F). The defect in Mec1 signaling
after MMS treatment was most apparent at early time points
after release into S phase (Fig. 5G). For example, at the 60-min
time point in the presence of either 0.005% or 0.01% MMS
both the phosphorylation-dependent shift of Rad53 and Rad53
kinase activity are significantly reduced in the ddc2?N strain
compared to DDC2 results (Fig. 5G). However, at later time
points (90 min), cells expressing Ddc2?N showed considerable
Rad53 activation whereas ?ddc2 cells did not (Fig. 5G). These
defects at early time points were not due to a difference in the
results of release of yeast from alpha factor arrest, since all
strains released equivalently. Taken together, these results sug-
gest that Ddc2-Rfa1 binding and localization to damage sites is
required for optimal checkpoint activation after exposure to
TopBP1-dependent ATR activation can occur independently
of RPA. TopBP1 was recently shown to bind and activate ATR
(28). This activation activity was localized to a small fragment
of TopBP1 between two BRCT repeat domains. These authors
also found that TopBP1 binding and activation of xATR re-
quires xATRIP. We confirmed that TopBP1 activates ATR-
ATRIP complexes in an ATRIP-dependent manner (Fig. 6
and data not shown). To determine whether the ATRIP CRD
influences TopBP1 activation of ATR, we purified either wild-
type ATR-ATRIP complexes or ATR-ATRIP?N complexes.
Addition of the TopBP1 fragment but not of an equivalent
fragment containing an inactivating mutation (W1145R) to
ATR-ATRIP complexes stimulated ATR activity toward sub-
strates in an immune complex kinase reaction (Fig. 6A). Ac-
tivation of the ATR-ATRIP?N complex upon the addition of
TopBP1 was equal to the activation of ATR-ATRIP (Fig. 6A).
These findings are consistent with those of Kumagai et al., who
found that xTopBP1 stimulates activation of xATR-xATRIP
complexes containing a xATRIP protein lacking the N termi-
nus (28). Therefore, TopBP1-dependent ATR activation does
not require the ATRIP CRD.
We next assayed whether RPA or RPA-ssDNA influences
ATR activity or TopBP1-dependent ATR activation. Addition
of TopBP1 to ATR-ATRIP stimulated ATR kinase activity
(Fig. 6B). In contrast, addition of RPA (data not shown) or
RPA-ssDNA to ATR kinase assays failed to stimulate ATR
activity (Fig. 6B). RPA-ssDNA also had no influence on
TopBP1 activation of ATR (Fig. 6B). RPA32 phosphorylation
by ATR is stimulated by TopBP1. In addition, we also ob-
served significant phosphorylation of the TopBP1 fragment,
ATRIP, and ATR in these experiments. However, in contrast
to the results seen with other proteins added to the kinase
assay, the amount of autophosphorylation on the ATR-ATRIP
complex was not altered significantly by the addition of the
TopBP1 fragment. These results suggest that RPA-ssDNA
binding to ATR-ATRIP does not influence the kinase activity
of ATR. Furthermore, the function of ATRIP required to
promote TopBP1-dependent activation of ATR can be sepa-
rated from its RPA binding activity. However, the results do
not exclude the possibility that specific RPA-DNA structures
found in cells might regulate kinase activity.
To confirm these results in cells, a GFP-TopBP1 fragment
containing the region that activates ATR was transfected into
human cells. The cells were engineered to stably express
siRNA-resistant wild-type ATRIP, ATRIP?N, or an empty
vector and were transfected with the ATRIP siRNA prior to
GFP-TopBP1 transfection. Depletion of endogenous ATRIP
by siRNA transfection in these cells is approximately 80% (3).
Twenty-four hours after transfection of GFP-TopBP1, cells
were fixed and stained for a marker of ATR activation
(?H2AX). Overexpression of GFP-TopBP1 in cells containing
wild-type ATRIP or ATRIP?N caused phosphorylation of
H2AX throughout the chromatin (not in distinct foci, as would
be observable in response to a DNA-damaging agent) (Fig.
6C). However, both the intensity of phosphorylation and the
number of cells containing phosphorylated H2AX were greatly
reduced in cells depleted of ATRIP, indicating that this result
was due to ATR-ATRIP signaling (Fig. 6C and D). These
results confirm that TopBP1 can activate ATR in cells when
highly overexpressed even when ATR-ATRIP complexes lack
the RPA binding domain and fail to localize to specific sites of
DNA damage or replication stress. The overexpression of the
TopBP1 fragment likely bypasses the regulation of TopBP1-
dependent ATR activation that exists under physiological con-
A checkpoint protein recruitment domain (CRD) has been
identified in the N terminus of ATRIP and Ddc2. This domain
binds directly to RPA70N, recruits ATR-ATRIP/Mec1-Ddc2
complexes to sites of DNA damage, and promotes ATR-de-
pendent checkpoint signaling in response to MMS. These find-
ings are consistent with those of Kim et al., who reported that
an N-terminal domain of Xenopus ATRIP is required for bind-
ing to RPA (25). RPA is a modular protein, and it often makes
more than one contact with its interacting partners. Indeed,
Namiki and Zou identified three large regions of ATRIP that
may interact with RPA-ssDNA (37). Since no functional data
were reported, additional experiments will be required to de-
fine and study the function of any other ATRIP surfaces that
make direct contacts with RPA subunits. However, our data
indicate that the N-terminal CRD domains of ATRIP and
Ddc2 are required for the stable binding of ATRIP/Ddc2 to
RPA and are necessary for retention of ATR-ATRIP/Mec1-
Ddc2 at sites of DNA damage in cells.
A model of the interaction of RPA70N with a conserved
ATRIP peptide within the CRD was generated using NMR
data and molecular modeling from the crystal structure of a
p53 peptide bound to RPA70N. The model predicts that acidic
ATRIP residues (D58 and D59) make direct contacts with
basic RPA70N residues (R41 and K88) in the basic cleft of the
RPA70N OB fold domain. All of these amino acids are highly
conserved. As predicted by this model, mutations reversing the
charges on the equivalent aspartic acid residues in Ddc2
(D12K and D13K) abrogate binding to Rfa1. Interestingly, the
well-characterized rfa1-t11 mutant, which is known to be rep-
lication competent but DNA-damage-response deficient, con-
tains a single charge-reversal mutation at K45, the residue
VOL. 27, 2007FUNCTIONS OF RPA AND TOPBP1 IN ATR-ATRIP SIGNALING 3375
equivalent to R41 in human RPA (49). Indeed, as our model
would predict, rfa-t11 is deficient in recruiting Ddc2 to double-
strand breaks (23, 52) and in binding Ddc2 (H. L. Ball, unpub-
lished data). The rfa-t11 mutant is also recombination defi-
cient, suggesting that this basic cleft in RPA70N may be a key
ligand in DNA damage responses (44, 49). It will be interesting
to determine whether other DNA damage response proteins
also contain acidic helices that bind within this cleft of
RPA70N. It is also noteworthy that an ATR phosphorylation
site (S68) is located within the ATRIP CRD just downstream
of the acidic peptide that binds to the RPA basic cleft (21).
Moreover, RPA70N appears to interact with the RPA32 N
terminus when it is phosphorylated by checkpoint kinases (6).
Therefore, phosphorylation of either ATRIP or RPA may be a
means to regulate the ATR-RPA interaction.
The phenotypic consequences of disrupting the ATRIP
CRD-RPA70 interaction are similar in human and yeast cells.
In contrast to ATRIP or Ddc2 loss of function, cells containing
mutations that disrupt the CRD are only mildly sensitive to
DNA-damaging agents and partially compromised in check-
point signaling. In fact, the response to HU is nearly indistin-
guishable from wild-type results despite severe defects in
ATR-ATRIP/Mec1-Ddc2 localization. Functions of ATRIP in
addition to RPA binding are also critical for ATR signaling.
These functions include oligomerization (2, 20), ATR stabili-
zation (12), and an undefined activity important for TopBP1-
dependent activation of ATR.
The reason for the increased sensitivity of Ddc2 lacking the
CRD to damage that generates DNA adducts (MMS) com-
pared to depletion of nucleotides (HU) is unknown. Both types
of genotoxic stress activate Mec1 during replication and stall
replication forks (48). One potential explanation for this dif-
ference may be the amount of RPA-ssDNA present at various
types of DNA lesions. Mec1-Ddc2?N complexes may have
some residual association with Rfa1 and can still partially lo-
calize to double-strand breaks. Perhaps there is more RPA-
ssDNA at an HU-stalled fork than at an MMS-induced lesion,
increasing the requirement for the Ddc2 CRD at the MMS
lesion. Alternatively, the recruitment and activation mecha-
nisms of ATR-ATRIP and Mec1-Ddc2 at MMS or HU lesions
may be different. Accumulating evidence suggests that addi-
tional protein-protein and protein-DNA interactions other
than the ATRIP-RPA interaction may help recruit ATR-
ATRIP to DNA lesions (8, 50). Also, Ddc2 contains a DNA
end-binding activity localized to a region C terminal to the
predicted coiled-coil domain (42). Perhaps these alternative
modes of ATR-ATRIP/Mec1-Ddc2 recruitment function dif-
ferently at HU and MMS lesions.
Consistent with the report by Kumagai and coworkers, we
have found that TopBP1 activates ATR and that TopBP1-
dependent activation of ATR is ATRIP dependent and does
not require the ATRIP CRD (28). RPA-ssDNA, in contrast,
does not stimulate ATR kinase activity in immune complex in
vitro kinase reactions, and TopBP1-dependent activation of
ATR is not altered by adding RPA or RPA-ssDNA to the
kinase reaction. These results suggest that TopBP1-dependent
ATR activation can be separated from ATRIP-RPA binding.
The affinity of TopBP1 for ATR-ATRIP is weak and difficult to
detect by coimmunoprecipitations (28). The accumulation of
ATR-ATRIP and TopBP1 at sites of damage may facilitate
this low-affinity interaction by increasing the local concentra-
tion of these proteins.
Taken together, these data support a multistep model pro-
posed by Dunphy and colleagues (27) for the activation of
ATR checkpoint signaling. ATR recruitment to sites of DNA
damage and replication stress occurs in part through a direct
interaction between the ATRIP CRD and RPA70N. TopBP1
is recruited independently through an interaction with Rad9
(16, 18, 35, 46). The assembly of ATR-ATRIP and TopBP1 at
the lesion facilitates TopBP1-dependent ATR activation and,
in turn, phosphorylation of ATR substrates. Accessory pro-
teins such as claspin are also required for phosphorylation of
specific substrates (26, 31). Localization may also serve to bring
ATR to the vicinity of key substrates involved in fork stabili-
zation or other aspects of checkpoint regulation. Within this
model, ATRIP is a key ATR regulator since it promotes both
the localization and activation of ATR. The model suggests
that ATR localization to a damage site precedes its activation.
However, it remains possible that ATR can be activated with-
out localization. Indeed, when TopBP1 is highly overexpressed
it activates ATR throughout the nucleus in the absence of a
DNA lesion (Fig. 6C). Furthermore, stable retention of ATR
at a damage site is not required for ATR activation, at least,
not in response to relatively high doses of UV (3). Thus,
further experiments are required to definitively determine
whether localization must precede activation.
ATR, ATM, and other checkpoint signaling pathways are
activated by many cancer therapies and regulate the cellular
outcomes of these treatments. Disruption of ATR, ATM, and
DNA-PK kinases sensitizes cells to radiation and chemother-
apy. Since mutations in DNA repair and DNA-damage re-
sponse pathways are common in cancer cells, these cells are
particularly sensitive to disruption of additional pathways. This
rationale has driven the development of small molecule inhib-
itors of DNA damage-responsive kinases for use as chemo- or
radio-sensitizing agents (22, 32). Thus far, specific inhibitors of
ATM and DNA-PK kinases have been developed, built around
competitive inhibition by binding of ATP analogues (19). How-
ever, specific inhibitors of the ATR kinase have not been
found. Unique properties of ATR, such as its requirement for
ATRIP, may provide an alternative means of disrupting ATR
signaling. Hence, the molecular characterization of ATRIP
structure and function as described here may provide a
useful starting point for the development of an ATR-targeted
We thank William Dunphy, Steve Jackson, Mike Resnick, Stephen
Elledge, Steven Brill, Tony Weil, and Jim Haber for reagents. We
thank Cheryl Arrowsmith for reagents and RPA70N NMR assign-
ments and Susan M. Meyn, Marie-Eve Chagot, and Kristian Kaufmann
for assistance in preparation of peptide and protein samples and ho-
This work was supported by grants from the National Cancer Insti-
tute (R01CA102729 to D.C.) and the National Institute of General
Medical Sciences (W.J.C.). D.C. is also supported by the Pew Scholars
Program in the Biological Sciences, sponsored by the Pew Charitable
Trusts. H.L.B. is supported by a Department of Defense predoctoral
fellowship and M.R.E. by an institutional training grant from the
National Institute of Environmental Health Sciences. Support for fa-
cilities was provided by grants to the Vanderbilt-Ingram Cancer Center
(National Cancer Institute) and the Vanderbilt Center in Molecular
3376 BALL ET AL.MOL. CELL. BIOL.
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