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.
Toxicology (National Institute of Environmental Health Sciences grant
1. Amberg, D. C., D. J. Burke, and J. E. Strathern. 2005. Methods in yeast
genetics: a Cold Spring Harbor laboratory course manual. Cold Spring Har-
bor Laboratory Press, Cold Spring Harbor, NY.
2. Ball, H. L., and D. Cortez. 2005. ATRIP oligomerization is required for
ATR-dependent checkpoint signaling. J. Biol. Chem. 280:31390–31396.
3. Ball, H. L., J. S. Myers, and D. Cortez. 2005. ATRIP binding to RPA-ssDNA
promotes ATR-ATRIP localization but is dispensable for Chk1 phosphory-
lation. Mol. Biol. Cell 16:2372–2381.
4. Bermudez, V. P., L. A. Lindsey-Boltz, A. J. Cesare, Y. Maniwa, J. D. Griffith,
J. Hurwitz, and A. Sancar. 2003. Loading of the human 9-1-1 checkpoint
complex onto DNA by the checkpoint clamp loader hRad17-replication
factor C complex in vitro. Proc. Natl. Acad. Sci. USA 100:1633–1638.
5. Binz, S. K., Y. Lao, D. F. Lowry, and M. S. Wold. 2003. The phosphorylation
domain of the 32-kDa subunit of replication protein A (RPA) modulates
RPA-DNA interactions. Evidence for an intersubunit interaction. J. Biol.
6. Binz, S. K., A. M. Sheehan, and M. S. Wold. 2004. Replication protein A
phosphorylation and the cellular response to DNA damage. DNA Repair
7. Bochkareva, E., L. Kaustov, A. Ayed, G. S. Yi, Y. Lu, A. Pineda-Lucena, J. C.
Liao, A. L. Okorokov, J. Milner, C. H. Arrowsmith, and A. Bochkarev. 2005.
Single-stranded DNA mimicry in the p53 transactivation domain interaction
with replication protein A. Proc. Natl. Acad. Sci. USA 102:15412–15417.
8. Bomgarden, R. D., D. Yean, M. C. Yee, and K. A. Cimprich. 2004. A novel
protein activity mediates DNA binding of an ATR-ATRIP complex. J. Biol.
9. Byun, T. S., M. Pacek, M. C. Yee, J. C. Walter, and K. A. Cimprich. 2005.
Functional uncoupling of MCM helicase and DNA polymerase activities
activates the ATR-dependent checkpoint. Genes Dev. 19:1040–1052.
10. Collins, I., and M. D. Garrett. 2005. Targeting the cell division cycle in
cancer: CDK and cell cycle checkpoint kinase inhibitors. Curr. Opin. Phar-
11. Cortez, D. 2005. Unwind and slow down: checkpoint activation by helicase
and polymerase uncoupling. Genes Dev. 19:1007–1012.
12. Cortez, D., S. Guntuku, J. Qin, and S. J. Elledge. 2001. ATR and ATRIP:
partners in checkpoint signaling. Science 294:1713–1716.
13. Edwards, R. J., N. J. Bentley, and A. M. Carr. 1999. A Rad3-Rad26 complex
responds to DNA damage independently of other checkpoint proteins. Nat.
Cell Biol. 1:393–398.
14. Ellison, V., and B. Stillman. 2003. Biochemical characterization of DNA
damage checkpoint complexes: clamp loader and clamp complexes with
specificity for 5? recessed DNA. PLoS Biol. 1:E33.
15. Falck, J., J. Coates, and S. P. Jackson. 2005. Conserved modes of recruit-
ment of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature
16. Furuya, K., M. Poitelea, L. Guo, T. Caspari, and A. M. Carr. 2004. Chk1
activation requires Rad9 S/TQ-site phosphorylation to promote association
with C-terminal BRCT domains of Rad4TOPBP1. Genes Dev. 18:1154–
17. Ghaemmaghami, S., W. K. Huh, K. Bower, R. W. Howson, A. Belle, N.
Dephoure, E. K. O’Shea, and J. S. Weissman. 2003. Global analysis of
protein expression in yeast. Nature 425:737–741.
18. Greer, D. A., B. D. Besley, K. B. Kennedy, and S. Davey. 2003. hRad9 rapidly
binds DNA containing double-strand breaks and is required for damage-
dependent topoisomerase II beta binding protein 1 focus formation. Cancer
19. Hickson, I., Y. Zhao, C. J. Richardson, S. J. Green, N. M. Martin, A. I. Orr,
P. M. Reaper, S. P. Jackson, N. J. Curtin, and G. C. Smith. 2004. Identifi-
cation and characterization of a novel and specific inhibitor of the ataxia-
telangiectasia mutated kinase ATM. Cancer Res. 64:9152–9159.
20. Itakura, E., I. Sawada, and A. Matsuura. 2005. Dimerization of the ATRIP
protein through the coiled-coil motif and its implication to the maintenance
of stalled replication forks. Mol. Biol. Cell 16:5551–5562.
21. Itakura, E., K. Umeda, E. Sekoguchi, H. Takata, M. Ohsumi, and A. Matsuura.
2004. ATR-dependent phosphorylation of ATRIP in response to genotoxic
stress. Biochem. Biophys. Res. Commun. 323:1197–1202.
22. Kaelin, W. G., Jr. 2005. The concept of synthetic lethality in the context of
anticancer therapy. Nat. Rev. Cancer 5:689–698.
23. Kanoh, Y., K. Tamai, and K. Shirahige. 2006. Different requirements for the
association of ATR-ATRIP and 9-1-1 to the stalled replication forks. Gene
24. Kawabe, T. 2004. G2 checkpoint abrogators as anticancer drugs. Mol. Cancer
25. Kim, S. M., A. Kumagai, J. Lee, and W. G. Dunphy. 2005. Phosphorylation
of Chk1 by ATM- and Rad3-related (ATR) in xenopus egg extracts requires
binding of ATRIP to ATR but not the stable DNA-binding or coiled-coil
domains of ATRIP. J. Biol. Chem. 280:38355–38364.
26. Kumagai, A., and W. G. Dunphy. 2000. Claspin, a novel protein required for
the activation of Chk1 during a DNA replication checkpoint response in
Xenopus egg extracts. Mol. Cell 6:839–849.
27. Kumagai, A., and W. G. Dunphy. 2006. How cells activate ATR. Cell Cycle
28. Kumagai, A., J. Lee, H. Y. Yoo, and W. G. Dunphy. 2006. TopBP1 activates
the ATR-ATRIP complex. Cell 124:943–955.
29. Lee, S. E., J. K. Moore, A. Holmes, K. Umezu, R. D. Kolodner, and J. E.
Haber. 1998. Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate
adaptation to G2/M arrest after DNA damage. Cell 94:399–409.
30. Lee, S. E., A. Pellicioli, M. B. Vaze, N. Sugawara, A. Malkova, M. Foiani, and
J. E. Haber. 2003. Yeast Rad52 and Rad51 recombination proteins define a
second pathway of DNA damage assessment in response to a single double-
strand break. Mol. Cell. Biol. 23:8913–8923.
31. Liu, S., S. Bekker-Jensen, N. Mailand, C. Lukas, J. Bartek, and J. Lukas.
2006. Claspin operates downstream of TopBP1 to direct ATR signaling
towards Chk1 activation. Mol. Cell. Biol. 26:6056–6064.
32. Lord, C. J., M. D. Garrett, and A. Ashworth. 2006. Targeting the double-
strand DNA break repair pathway as a therapeutic strategy. Clin. Cancer
33. Lukas, J., C. Lukas, and J. Bartek. 2004. Mammalian cell cycle checkpoints:
signalling pathways and their organization in space and time. DNA Repair
34. Luo, Y., and J. D. Leverson. 2005. New opportunities in chemosensitization
and radiosensitization: modulating the DNA-damage response. Expert Rev.
Anticancer Ther. 5:333–342.
35. Ma ¨kiniemi, M., T. Hillukkala, J. Tuusa, K. Reini, M. Vaara, D. Huang, H.
Pospiech, I. Majuri, T. Westerling, T. P. Makela, and J. E. Syvaoja. 2001.
BRCT domain-containing protein TopBP1 functions in DNA replication
and damage response. J. Biol. Chem. 276:30399–30406.
36. Melo, J. A., J. Cohen, and D. P. Toczyski. 2001. Two checkpoint complexes
are independently recruited to sites of DNA damage in vivo. Genes Dev.
37. Namiki, Y., and L. Zou. 2006. ATRIP associates with replication protein
A-coated ssDNA through multiple interactions. Proc. Natl. Acad. Sci. USA
38. Paciotti, V., M. Clerici, G. Lucchini, and M. P. Longhese. 2000. The check-
point protein Ddc2, functionally related to S. pombe Rad26, interacts with
Mec1 and is regulated by Mec1-dependent phosphorylation in budding yeast.
Genes Dev. 14:2046–2059.
39. Pellicioli, A., C. Lucca, G. Liberi, F. Marini, M. Lopes, P. Plevani, A.
Romano, P. P. Di Fiore, and M. Foiani. 1999. Activation of Rad53 kinase in
response to DNA damage and its effect in modulating phosphorylation of the
lagging strand DNA polymerase. EMBO J. 18:6561–6572.
40. Rohl, C. A., C. E. Strauss, D. Chivian, and D. Baker. 2004. Modeling
structurally variable regions in homologous proteins with rosetta. Proteins
41. Rouse, J., and S. P. Jackson. 2000. LCD1: an essential gene involved in
checkpoint control and regulation of the MEC1 signalling pathway in Sac-
charomyces cerevisiae. EMBO J. 19:5801–5812.
42. Rouse, J., and S. P. Jackson. 2002. Lcd1p recruits Mec1p to DNA lesions in
vitro and in vivo. Mol. Cell 9:857–869.
43. Shechter, D., V. Costanzo, and J. Gautier. 2004. Regulation of DNA repli-
cation by ATR: signaling in response to DNA intermediates. DNA Repair
44. Soustelle, C., M. Vedel, R. Kolodner, and A. Nicolas. 2002. Replication
protein A is required for meiotic recombination in Saccharomyces cerevisiae.
45. Stauffer, M. E., and W. J. Chazin. 2004. Physical interaction between repli-
cation protein A and Rad51 promotes exchange on single-stranded DNA.
J. Biol. Chem. 279:25638–25645.
46. St. Onge, R. P., B. D. Besley, J. L. Pelley, and S. Davey. 2003. A role for the
phosphorylation of hRad9 in checkpoint signaling. J. Biol. Chem. 278:26620–
47. Storici, F., L. K. Lewis, and M. A. Resnick. 2001. In vivo site-directed
mutagenesis using oligonucleotides. Nat. Biotechnol. 19:773–776.
48. Tercero, J. A., M. P. Longhese, and J. F. Diffley. 2003. A central role for
DNA replication forks in checkpoint activation and response. Mol. Cell
49. Umezu, K., N. Sugawara, C. Chen, J. E. Haber, and R. D. Kolodner. 1998.
Genetic analysis of yeast RPA1 reveals its multiple functions in DNA me-
tabolism. Genetics 148:989–1005.
50. Unsal-Kac ¸maz, K., A. M. Makhov, J. D. Griffith, and A. Sancar. 2002.
Preferential binding of ATR protein to UV-damaged DNA. Proc. Natl.
Acad. Sci. USA 99:6673–6678.
51. Wakayama, T., T. Kondo, S. Ando, K. Matsumoto, and K. Sugimoto. 2001.
Pie1, a protein interacting with Mec1, controls cell growth and checkpoint
responses in Saccharomyces cerevisiae. Mol. Cell. Biol. 21:755–764.
52. Zou, L., and S. J. Elledge. 2003. Sensing DNA damage through ATRIP
recognition of RPA-ssDNA complexes. Science 300:1542–1548.
53. Zou, L., D. Liu, and S. J. Elledge. 2003. Replication protein A-mediated
recruitment and activation of Rad17 complexes. Proc. Natl. Acad. Sci. USA
VOL. 27, 2007FUNCTIONS OF RPA AND TOPBP1 IN ATR-ATRIP SIGNALING3377