Cdc45 Protein-Single-stranded DNA Interaction Is Important
for Stalling the Helicase during Replication Stress*□
Irina Bruck and Daniel L. Kaplan1
FromtheFloridaStateUniversityCollegeofMedicine,DepartmentofBiomedicalSciences,Tallahassee, Florida 32306
Background: Polymerase stalling is coupled with helicase stalling at a eukaryotic replication fork by an unknown
of the helicase from the polymerase.
Conclusion: Cdc45-ssDNA interaction is important during replication stress.
Significance: A new model explains how polymerase stalling is coupled with helicase stalling.
Replicative polymerase stalling is coordinated with replica-
tive helicase stalling in eukaryotes, but the mechanism underly-
helicase. We report here that Cdc45 from budding yeast binds
(ssDNA) and that 60mer ssDNA specifically disrupts the inter-
action between Cdc45 and Mcm2-7. We identified a mutant of
Cdc45 that does not bind to ssDNA. When this mutant of cdc45
is expressed in budding yeast cells exposed to hydroxyurea, cell
growth is severely inhibited, and excess RPA accumulates at or
near an origin. Chromatin immunoprecipitation suggests that
helicase movement is uncoupled from polymerase movement
for mutant cells exposed to hydroxyurea. These data suggest
case during replication stress.
DNA replication in eukaryotes is a highly regulated event
(1–5). Central to the activation of DNA replication is assembly
protein, the heterohexamer Mcm2-7 ring, and the tetrameric
GINS complex (6–8). This large helicase assembly is also
known as the CMG2(Cdc45?Mcm2-7?GINS) complex. The
of two S phase cellular kinases, the Dbf4-dependent kinase and
the cyclin-dependent kinase (9–13). The activity of Dbf4-de-
pendent kinase is specifically required for incorporation of
Cdc45 with Mcm2-7, whereas cyclin-dependent kinase is
required for the recruitment of GINS to replication origins
ATPase activity compared with Mcm2-7 helicase alone (15).
The CMG complex is a highly processive and active helicase
with high ATPase rates, whereas Mcm2-7 is a very poor
ATPase, and particular reaction conditions are required to
observe even weak activity in vitro (15–17). These data suggest
that Cdc45 and GINS are required for full activation of the
replication fork helicase (15, 16). Recent electron microscopy
data suggest that the binding of Cdc45 and GINS to Mcm2-7
may favor a closed-ring conformation of Mcm2-7 and that this
and unwinding activity (18). Cdc45, Mcm2-7, and GINS travel
with one another away from replication origins (7, 19), and
recent data show that the CMG complex surrounds a single
strand of DNA during helicase unwinding (20).
Cdc45 binds to Sld3 during much of the cell cycle (21). Fur-
important for delivering Cdc45 to the Mcm2-7 complex, and
this event may require the prior activity of Dbf4-dependent
kinase (21–24). However, Sld3 does not travel with the replica-
tion fork, unlike Cdc45 (25). In vitro, interaction of Cdc45 with
Sld3 is disrupted by GINS, suggesting an in vivo mechanism to
dissociate Cdc45 from Sld3 (22). Cdc45 also binds directly to
Escherichia coli nuclease RecJ (26). The functional importance
of Cdc45 homology with RecJ has not yet been elucidated.
Moreover, the function of the C-terminal region of Cdc45 has
not yet been defined.
During episodes of DNA polymerase stalling, a coordinated
during DNA polymerase stalling events, and RPA-coated
ssDNA acts as a signal to trigger the DNA damage response
(27–31). The cascade of events involved during the DNA dam-
age response has been characterized previously (27–31). One
bition of DNA replication initiation activity (27–32).
When the DNA polymerases are induced to stall by
unwound by the replication fork helicase ahead of the poly-
34). Thus, polymerase stalling is coupled with helicase stalling
in eukaryotic cells in a manner that does not depend upon the
* This work was supported by National Science Foundation Grant 1121534
(to D. L. K.).
SThis article contains supplemental Figs. S1–S4 and References.
Sciences, Florida State University College of Medicine, Tallahassee, FL
32306. Tel.: 850-645-0237; Fax: 850-645-5781; E-mail: daniel.kaplan@
2The abbreviations used are: CMG, Cdc45?Mcm2-7?GINS; ssDNA, single-
stranded DNA; HU, hydroxyurea; dsDNA, double-stranded DNA; MMS,
methyl methanesulfonate; GINS, Go-Ichi-Ni-San; 5,1,2,3, Sld5, Psf1, Psf2,
Psf3; RPA, Replication Protein A.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 11, pp. 7550–7563, March 15, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
by guest on September 23, 2015
DNA damage response. However, thus far, little is understood
about how DNA polymerase stalling is directly coordinated
with DNA helicase inhibition.
genomic ssDNA sequences. We also report that ssDNA specif-
ically disrupts the interaction between Cdc45 and Mcm2-7,
suggesting a mechanism to inhibit replication fork helicase
binding activity, and we identified a mutant of Cdc45 (Cdc45-
M268) that does not bind to ssDNA. The Cdc45-M268 mutant
exhibits wild-type binding to Sld3, Mcm2-7, and GINS. When
the cdc45-m268 mutant is expressed in budding yeast cells, cell
growth is severely inhibited in the presence of hydroxyurea or
methyl methanesulfonate. cdc45-m268 cells exposed to
hydroxyurea also exhibit increased RPA accumulation near an
origin. ChIP data suggest that upon exposure to hydroxyurea,
the cdc45-m268 mutant cells have helicase movement uncou-
pled from polymerase movement. These data suggest that
Cdc45-ssDNA interaction is important for stalling the helicase
during replication stress.
Proteins—Proteins were purified as described (14, 22). GST-
Cdc45, PKA-Cdc45, native Cdc45, GST-Sld3, PKA-Sld3, GST-
Mcm2-7, PKA-Mcm2-7, and PKA-GINS were purified as
described. GST-Cdc45 (fragments and mutants) and PKA-
Cdc45 (fragments and mutants) were purified as the wild-type,
full-length Cdc45. Protein kinase A was a generous gift from
Susan Taylor. SDS/PAGE/Coomassie analysis of purified pro-
teins is shown in supplemental Fig. S1. ?-Factor peptide pher-
omone was obtained from Zymo Research (catalog no. Y1001).
Strains—The cdc45-td degron strain (YBH42, MATa
ade2-1 ura3-1 his3-11,15 trp1-1 leu2–3,112 can1–100, Gal:
UBR1(HIS3), cdc45-td (KanMX)) (35) was a generous gift of
Karim Labib (Paterson Institute for Cancer Research, Univer-
sity of Manchester, Manchester, UK). The degron strain was
transformed with the pRS415 plasmid (low copy) containing a
wild-type or mutant copy of cdc45 under control of its native
The wild-type or mutant CDC45 gene and native promoter
were PCR-amplified from linearized pRS415 containing wild-
type or mutant CDC45 to include a Leu marker and 5? and 3?
sequences to allow for homologous recombination into the
haploid -Leu strain (BY4741). Transformants were selected on
complete supplement mixture-Leu medium.
GST Pull-down—The GST pull-down reactions were per-
formed as described (37). GST pull-down reactions were per-
formed in a volume of 100 ?l and contained GST-tagged pro-
buffer (40 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA,
radiolabeled DNA or protein as described in each figure. Reac-
tions were incubated at room temperature for 1 h. Following
incubation, reactions were added to 40 ?l of prepared glutathi-
one-Sepharose and mixed gently. Binding of GST-tagged pro-
tein to the beads was performed for 20 min with gentle mixing
every few minutes. When the binding was complete, the beads
were allowed to settle, the supernatant was removed, and the
glutathione beads were washed twice with 0.5 ml GST-binding
buffer. After the last wash, 30 ?l of 5? SDS sample buffer was
Samples (20 ?l) were then analyzed by SDS-PAGE.
Biotin Pull-down—The biotin pull-down reaction were per-
formed as described (38). Biotinylated DNA conjugated to
streptavidin-agarose magnetic beads (Dynal) were incubated
for 5 min at 30 °C with various concentrations of radiolabeled
mM magnesium acetate, 10% glycerol, 40 ?g/ml BSA, 100 mM
NaCl, and 20 mM Tris-HCl (pH 7.5) in a final volume of 25 ?l.
After the 5-min incubation, the beads were collected at room
ing 0.1 mM EDTA, 0.2 mM DTT, 10 mM magnesium acetate,
tant was aspirated, and the beads were heated at 95 °C for 10
mM Tris-HCl, and 0.01% bromphenol blue. The reactions were
analyzed by SDS-PAGE. The gel was dried for 1 h at 80 °C and
exposed to a phosphorimaging screen for 1 h.
Fluorescence Anisotropy—Fluorescence anisotropy was per-
formed as described (38). Cdc45-binding to DNA was mea-
Cdc45 were incubated with 10 nM fluorescent DNA (5-Car-
boxyfluorescein) for 5 min in a solution containing 0.1 mM
EDTA, 0.2 mM DTT, 10 mM magnesium acetate, 10% glycerol,
tation and emission wavelengths of 495 and 538 nm, respec-
tively. The data were plotted as change in anisotropy versus
protein concentration, and dissociation constants (Kd) were
derived by fitting the data with the equation y ? m0/(m0 ?
m1), where m1 ? Kd.
N terminus (Cdc45, Sld3, Mcm2-7 (Mcm3), and GINS (Psf1))
20 ?M of protein kinase A-tagged protein in kinase reaction
buffer (5 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 1 mM DTT, 500
?M ATP, 500 ?Ci [?-32P]ATP) containing 5 ?g of protein
kinase A. Reactions were incubated for 1 h at 30 °C.
EMSA—1 nM radiolabeled DNA (single-stranded polymers
of dT of various lengths) was mixed with various concentra-
tions of protein (as detailed in Fig. 2) in 1? EMSA buffer (con-
taining 0.1 mM EDTA, 0.2 mM DTT, 10% glycerol, 40 mg/ml
BSA, and 20 mM Tris-HCl (pH 7.5)) for 5 min at 30 °C. 4%
Glycerol and 0.01% bromphenol blue were then added to the
solution, and the reaction was analyzed by a 10% native poly-
acrylamide gel in 0.5 ? Tris-Borate-EDTA buffer. The gel was
for 1 h.
Size Exclusion Chromatography—Size exclusion chromatog-
raphy was performed as described (22). Unlabeled protein was
mixed with radiolabeled protein as described in the Figs. 3–5
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in column buffer (50 mM Tris (pH 7.5), 100 mM NaCl, 1 mM
24 ml Superose 6 (GE Life Sciences) size exclusion chromatog-
raphy in the same column buffer. The radiolabeled protein
from each 250 ?l fraction was then quantified and plotted.
Serial Dilution Analysis—Serial dilution was performed as
described (37). For degron experiments, overnight culture
was transferred into YPGal medium and incubated for 1 h at
24 °C. Then, 10-fold serial dilutions were performed on the
described previously. For allelic replacement, cells were grown
overnight and plated onto the indicated media at 30 °C.
Fluorescence-activated Cell Sorting—FACS was performed
overnight at 30 °C. For G1arrest and release experiments, 6 ?
106cells/ml were transferred into yeast-peptone-dextrose
medium and treated with ?-factor (Zymo Research) for 3 h at
nase (Calbiochem) to fresh YPD. Cells were then incubated
further for the indicated time. Cell cycle progression was fol-
In Situ Autophosphorylation Assay—An in situ autophos-
phorylation assay was performed as described (39). Cells were
grown in YPD at 30 °C and arrested with ?-factor. Cells were
then released into 0.2 M HU for 90 min, and samples were col-
lected and analyzed. Protein samples were prepared and trans-
ferred by immunoblotting onto PVDF. The membrane was
EDTA, 50 mM DTT, 7 M guanidine HCl, 1 h at 24 °C), washed
twice with TBS, and renatured (10 mM Tris HCl (pH 7.5), 140
was washed with 30 mM Tris HCl (pH 7.5) for 1 h and then
equilibrated in kinase buffer (40 mM Hepes NaOH (pH 8.0), 1
mM DTT, 0.1 mM EGTA, 20 mM MnCl2, 100 ?M sodium
orthovanadate) for 30 min at room temperature. Fresh kinase
buffer supplemented with 10 ?Ci/ml [?-32P]ATP was added to
blot was washed extensively (10 min each) twice with 30 mM
Nonidet P-40, once with 30 mM Tris HCl (pH 7.5), once with
1 M potassium hydroxide, once with H2O, once with 10% TCA,
and once with H2O. The membrane was dried and analyzed
using a phosphorimager.
Antibodies—Antibodies directed against RPA (Pierce, cata-
log no. MA1-25889), Rad53 (Santa Cruz Biotechnology, Inc.,
catalog no. SC-6752) were purchased. Open Biosystems pro-
bilized antigen to remove nonspecific antibodies. The specific-
ity of each antibody was analyzed by Western blot analysis of
mental Fig. S4.
ChIP—For G1arrest and release experiments, 6 ? 106
and addition of 50 ?g/ml Pronase (Calbiochem) to fresh YPD.
immunoprecipitation was performed as described (40, 41),
the PCR reaction to quantify the amplified product (42). Form-
beater. DNA was fragmented by sonication (Branson 450).
Invitrogen, catalog no. 100.02D) were added to the cleared
lysate to immunoprecipitate the DNA. Immunoprecipitates
were washed extensively to remove nonspecific DNA. Eluted
DNA was then subjected to PCR analysis using primers
directed against ARS305 or ?4 kb upstream of ARS305 as
tified by phosphorimaging and normalized by a reference
replacing the immunoprecipitate.
Cdc45 Binds Single-stranded DNA with Little Sequence
Specificity—Cdc45 is part of the replication fork helicase
assembly in eukaryotes, and we postulated that Cdc45 might
bind directly to ssDNA as part of its function. We purified
Cdc45, Sld3, Mcm2-7, and GINS to homogeneity as described
previously (Coomassie-stained gels are shown in supplemental
Fig. S1) (22, 43, 44). Because Cdc45 initially binds at a replica-
tion origin and then travels away from the origin (8), we inves-
tigated budding yeast Cdc45 interaction with ssDNA and
sequences positioned at the origin ARS1 and non-origin
plemental Fig. S2A). 80-mer ssDNA was fluorescently labeled
and tested for interaction with Cdc45 by fluorescence anisot-
ropy (Fig. 1A). Using this technique, we found that Cdc45
bound to 80-mer ARS1 ssDNA and 80-mer ssDNA positioned
site binding equation and found that the dissociation constant
for Cdc45 binding to these 80-mer ssDNA sequences varied
from 59 ? 5 nM to 79 ? 6 nM. However, Cdc45 bound very
at a region positioned 1 kb upstream, suggesting that Cdc45-
DNA interaction is specific for ssDNA (Fig. 1A).
To determine whetherCdc45 binding to ssDNA was specific
for ARS1 and neighboring sequences, we also examined the
interaction between Cdc45 and the early origin ARS305 and
sequences positioned 0.5 kb and 1 kb downstream of ARS305
sequences with a dissociation constant that varied from 54 ? 4
nM to 74 ? 5 nM (supplemental Fig. S3B). Binding of Cdc45 to
dsDNA encompassing the same regions was substantially
weaker (supplemental Fig. S3B). We confirmed the interaction
between Cdc45 and the 80-mer ssDNA sequences using GST-
Cdc45 and radiolabeled ssDNA (supplemental Fig. S3C). In
summary, the data suggest that Cdc45 binds to 80-mer ssDNA
7552 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER11•MARCH15,2013
by guest on September 23, 2015
flow method. J. Mol. Biol. 343, 83–99
51. Ribeck, N., Kaplan, D. L., Bruck, I., and Saleh, O. A. (2010) DnaB helicase
52. Hashimoto, Y., Puddu, F., and Costanzo, V. (2012) RAD51- and MRE11-
dependent reassembly of uncoupled CMG helicase complex at collapsed
replication forks. Nat. Struct. Mol. Biol. 19, 17–24
Functional uncoupling of MCM helicase and DNA polymerase activities
activates the ATR-dependent checkpoint. Genes Dev. 19, 1040–1052
54. Branzei, D., and Foiani, M. (2009) The checkpoint response to replication
stress. DNA Repair 8, 1038–1046
55. Kastan, M. B., and Bartek, J. (2004) Cell-cycle checkpoints and cancer.
Nature 432, 316–323
56. Cimprich, K. A., and Cortez, D. (2008) ATR. An essential regulator of
genome integrity. Nat. Rev. Mol. Cell Biol. 9, 616–627
by guest on September 23, 2015
Irina Bruck and Daniel L. Kaplan
2013, 288:7550-7563. J. Biol. Chem.
Helicase during Replication Stress
Interaction Is Important for Stalling the
Cdc45 Protein-Single-stranded DNA
DNA and Chromosomes:
doi: 10.1074/jbc.M112.440941 originally published online February 4, 2013
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