INVESTIGATION
The Role of Dbf4-Dependent Protein Kinase in DNA
Polymerase z-Dependent Mutagenesis in
Saccharomyces cerevisiae
Luis N. Brandão,*,1,2 Rebecca Ferguson,1,* Irma Santoro,†,3 Sue Jinks-Robertson,†,‡ and Robert A. Sclafani*
*Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045,
of Biology, Emory University, Atlanta, Georgia 30322, and ‡Department of Molecular Genetics and Microbiology,
Duke University Medical Center, Durham, North Carolina 27710
†Department
ABSTRACT The yeast Dbf4-dependent kinase (DDK) (composed of Dbf4 and Cdc7 subunits) is an essential, conserved Ser/Thr protein
kinase that regulates multiple processes in the cell, including DNA replication, recombination and induced mutagenesis. Only DDK
substrates important for replication and recombination have been identified. Consequently, the mechanism by which DDK regulates
mutagenesis is unknown. The yeast mcm5-bob1 mutation that bypasses DDK’s essential role in DNA replication was used here to
examine whether loss of DDK affects spontaneous as well as induced mutagenesis. Using the sensitive lys2DA746 frameshift reversion
assay, we show DDK is required to generate “complex” spontaneous mutations, which are a hallmark of the Polz translesion synthesis
DNA polymerase. DDK co-immunoprecipitated with the Rev7 regulatory, but not with the Rev3 polymerase subunit of Polz. Conversely,
Rev7 bound mainly to the Cdc7 kinase subunit and not to Dbf4. The Rev7 subunit of Polz may be regulated by DDK phosphorylation as
immunoprecipitates of yeast Cdc7 and also recombinant Xenopus DDK phosphorylated GST-Rev7 in vitro. In addition to promoting Polzdependent mutagenesis, DDK was also important for generating Polz-independent large deletions that revert the lys2DA746 allele. The
decrease in large deletions observed in the absence of DDK likely results from an increase in the rate of replication fork restart after an
encounter with spontaneous DNA damage. Finally, nonepistatic, additive/synergistic UV sensitivity was observed in cdc7D pol32D and
cdc7D pol30-K127R,K164R double mutants, suggesting that DDK may regulate Rev7 protein during postreplication “gap filling” rather
than during “polymerase switching” by ubiquitinated and sumoylated modified Pol30 (PCNA) and Pol32.
K
NOWLEDGE of how mutations are produced is important for understanding genetic variability in populations
and the process of evolution. Cells have evolved sophisticated molecular mechanisms for maintaining the integrity
of the genome. Most DNA repair mechanisms have high
fidelity and prevent mutations by removing damaged DNA
and replacing the resulting gap using the undamaged, complementary strand as a template (Waters et al. 2009; Boiteux
and Jinks-Robertson 2013). If not repaired, some lesions
Copyright © 2014 by the Genetics Society of America
doi: 10.1534/genetics.114.165308
Manuscript received April 14, 2014; accepted for publication May 23, 2014; published
Early Online May 28, 2014.
1
These authors contributed equally to this work.
2
Present address: Gevo, 345 Inverness Dr. S., Bldg. C, Ste. 310, Englewood, CO
80112.
3
Present address: Department of Biology, 7300 Reinhardt Circle, Reinhardt
University, Waleska, GA 30183.
4
Corresponding author: Department of Biochemistry and Molecular Genetics,
University of Colorado, 12801 E. 17th Ave, MS8101, Aurora, CO 80045.
E-mail: [email protected]
block the replicative DNA polymerases (Pols a, d, and e)
during S phase and require bypass by an alternative errorfree or error-prone mechanism. Error-free bypass usually
involves a template switch to the undamaged sister chromatid, while error-prone bypass uses translesion synthesis
(TLS) DNA polymerases to synthesize new DNA directly
across the lesion. There are at least 15 identified TLS polymerases in human cells but only three in the yeast Saccharomyces cerevisiae: Polz, Polh, and Rev1, which are encoded
by the REV3-REV7, RAD30, and REV1 genes, respectively
(Boiteux and Jinks-Robertson 2013). TLS polymerases have
low fidelity and processivity on undamaged DNA templates
and lack an associated exonuclease proofreading activity.
The misregulation or loss of TLS polymerases is known to
result in a number of human diseases, underscoring the
importance of this process. Patients suffering from a variant
form of xeroderma pigmentosum, for example, lack Polh
and have an increased risk for skin cancer (Kennedy and
D’Andrea 2006). In Fanconi anemia, another high-risk familial
Genetics, Vol. 197, 1111–1122 August 2014
1111
cancer syndrome, patients are TLS defective due to a failure
to recruit Rev1, a nontemplate directed deoxycytidyl terminal transferase, to the site of DNA damage (Kim et al.
2012).
In early studies by Kilbey (Njagi and Kilbey 1982) and
subsequent studies by our laboratory, induced mutagenesis in S.
cerevisiae was reduced in hypomorphic cdc7 mutants including
an inactive cdc7 kinase-dead (KD) mutant (Hollingsworth et al.
1992; Ostroff and Sclafani 1995; Pessoa-Brandão and Sclafani
2004). Dbf4-dependent kinase (DDK) is conserved from yeast
to humans and is composed of the Cdc7 kinase catalytic and
Dbf4 regulatory subunit. Dbf4 is low in G1 phase of the cell
cycle and required for kinase activity (Sclafani and Holzen
2007). DDK is a Ser/Thr protein kinase that regulates the
initiation of DNA replication by phosphorylating one or
more members of the Mcm2–7 DNA helicase (Sheu and
Stillman 2006; Randell et al. 2010; Sheu and Stillman
2010; Heller et al. 2011; reviewed in Tanaka and Araki
2013). Either mcm5 (mcm5-bob1) (Hardy et al. 1997) or
mcm4 (mcm4D74-174) (Sheu and Stillman 2010) mutations
can bypass the requirement for DDK during replication.
However, cdc7D mcm5-bob1, dbf4D mcm5-bob1, or cdc7D
dbf4D mcm5-bob1 strains are defective in other DDK functions, including the regulation of meiotic recombination
(Matos et al. 2008; Wan et al. 2008) and resistance to DNA
damaging agents such as UV and MMS (Pessoa-Brandão and
Sclafani 2004), suggesting the kinase interacts with and
phosphorylates additional substrates that promote mutagenesis and meiotic recombination (Sclafani 2000). In this report, we investigated the role of DDK in mutagenesis and
demonstrate DDK is required for spontaneous as well as induced Polz-dependent mutagenesis. Based on biochemical
analyses, we suggest DDK regulates mutagenesis by direct interaction with and phosphorylation of the Rev7 subunit of
Polz. Our results are important, given the conservation of
DDK’s role in TLS in human cells (Day et al. 2010; Yamada
et al. 2013), and are significant with regard to human disease
because overexpression of yeast DDK increases mutagenesis
(Sclafani et al. 1988; Sclafani and Jackson 1994), human
DDK overexpression is observed in many types of cancer cells
(Hess et al. 1998; Nambiar et al. 2007; Bonte et al. 2008;
Clarke et al. 2009; Kulkarni et al. 2009), and DDK is a therapeutic target in cancer patients (Rodriguez-Acebes et al. 2010).
Materials and Methods
Yeast strains, media, and plasmids
Yeast strains and plasmids used are listed in Table 1 and
Table 2, respectively. Yeast strains were grown in yeast extract/
peptone/dextrose (YPD) with 2% glucose or in synthetic
defined minimal media supplemented with appropriate amino
acids and 2% glucose (Pessoa-Brandão and Sclafani 2004).
Galactose induction of tagged genes for subsequent immunoprecipitation was as described (Oshiro et al. 1999). Briefly,
exponentially growing cells using raffinose as carbon source
1112
L. N. Brandão et al.
were induced by the addition galactose to 2% for 2 hr. Because
pSF2 has a copper-inducible promoter, pSF2-containing cells
were induced by the addition of 0.1 mM CuSO4 for 2 hr.
Genetic methods for yeast strain construction, tetrad
analysis, and transformation were as previously described
(Pessoa-Brandão and Sclafani 2004). Gene deletions marked
with the kanMX cassette were constructed by PCR amplification of an appropriate disruption cassette, transformation,
and subsequent selection with G418 (Winzeler et al. 1999).
The pol30-K127,K164R allele was introduced at the POL30
locus by two-step allele replacement using XbaI-digested plasmid 721 and verified by sequencing genomic DNA. The
mcm5-bob1-2 allele contains a CT-to-TC mutation that converts codon 83 from proline to leucine and creates a DdeI site.
This allele was introduced into strain SJR1177 by two-step
allele replacement using MluI-digested pRAS490, which contains mcm5-bob1-2 in pRS306 (Pessoa-Brandão and Sclafani
2004; Dohrmann and Sclafani 2006). The cdc7D::HIS3 allele
was introduced into RSY1183 by one-step allele replacement
using a 3-kb MluI–EcoRI fragment from pRS277-cdc7D::HIS3
(Jackson et al. 1993). Presence of the cdc7D::HIS3 allele was
verified by restriction digest and by loss of cdc7 genetic complementation. Strain YSS13 contains Myc-tagged Rev7 and
was obtained from Marco Muzi-Falconi (University of Milan,
Milan, Italy) (Sabbioneda et al. 2005).
pLPB60 encodes GST-tagged Rev7 and was constructed
by insertion of REV7 into the NcoI/BamHI sites of the
pYES263 expression vector (Melcher 2000). Plasmid pLPB46,
which inserts a FLAG (Sigma-Aldrich) epitope tag immediately after the initiating methionine of Pol30, was constructed by PCR-overlap mutagenesis (Ho et al. 1989).
Plasmid pBL211 was used as template for PCR using outside primers M13Fwd (59-TGTAAAACGACGGCCAGT-39)
and M13Rev (59-TCACACAGGAAACAGCTATGAC-39), complementary to the plasmid backbone, and internal primers
Pol30-FlagFwd (59-ATGgattacaaggatgacgacgataagTTAGAA
GCAAAATTTGAAGAAGC-39) and Pol30-FlagRev (59-cttatcg
tcgtcatccttgtaatcCATTTTTTCTCTCTTTTGACTGCG-39; lowercase
letters indicate the FLAG epitope tag). The resulting PCR
fragment, which includes the FLAG-Pol30 construct and
the POL30 promoter, was then digested with XhoI and
BamHI and cloned into pRS426. The FLAG-Pol30 gene
was functional as plasmid pLPB46 complemented a lethal
pol30D::kanMX4 allele.
All tagged constructs used in this study (Table 1 and Table
2) have been shown to function normally by genetic complementation: Cdc7-myc9 (Oshiro et al. 1999), HA-Dbf4 (Oshiro
et al. 1999), HA-Cdc7 (Hardy and Pautz 1996), GST-Mcm2
(Lei et al. 1997), GST-Rev1 and GST-Rev3 (Nelson et al.
1996), and REV7-myc13 (Sabbioneda et al. 2005). GST-Rev7
produced in this study was tested by complementation of
a rev7D::KanMX4 null mutant.
Mutagenesis assays
UV mutagenesis assays using the CAN1 forward mutation
system were as described previously (Hollingsworth et al.
Table 1 S. cerevisiae strains used in this study
Strains
RSY299
RSY466
P211
P235
SJR1177
RSY1183
RSY1190
RSY1224
YSS13
yLPB21
yLPB24
yLPB91
yLPB95
yLPB224
yLPB226
yLPB132
YJO235
RSY1445
yLPB163
Relevant genotype
Background/source or reference
MATa his3Δ1 leu2 trp1 ura3 can1 cyh2
MATa leu2 ura3 trp1 his7
MATa ura3 lys2 cyh2 his3 leu2 cdc7Δ::HIS3 mcm5-bob1-1
MATa ura3 lys2 cyh2 his3 leu2 cdc7Δ::HIS3 dbf4Δ::URA3 mcm5-bob1-1
MATa ade2-101oc his3D200 ura3DNco lys2DA746 rad1::hisG
MATa ade2-101oc his3D200 ura3DNco lys2DA746 rad1::hisG mcm5-bob1-2
MATa ade2-101oc his3D200 ura3DNco lys2DA746 rad1::hisG mcm5-bob1-2
cdc7D::HIS3
MATa ade2-101oc his3D200 ura3DNco lys2DA746 rad1::hisG mcm5-bob1-2
rev3D::kanMX4
MATa ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 RAD5 REV7-myc13::
kanMX4
MATa ura3 his3 leu2 cyh2 trp1 can1
MATa ura3 his3 leu2 cyh2 cdc7Δ::HIS3 mcm5-bob1-1
MATa ura3 his3 leu2 cyh2 trp1 can1 pol32Δ::kanMX4
MATa ura3 his3 leu2 cyh2 lys2 cdc7Δ::HIS3 mcm5-bob1-1 pol32Δ::kanMX4
MATa his3 leu2 trp1 ura3 can1 cyh2 pol30-K127R,K164R
MATa ura3 lys2 cyh2 his3 leu2 cdc7Δ1::HIS3+ mcm5-bob1-1 pol30-K127R,K164R
MATa ura3 leu2 trp1 his7 mcm5-bob1 cdc7(D163N)-myc9
MATa ura3 leu2 trp1 bar1::LEU2 CDC7-myc9
MATa ura3 leu2 his3 mcm5-bob1 cdc7Δ1::LEU2 dbf4D::URA3 Rev7-myc13 KanMX4
MATa ura3 leu2 his3 mcm5-bob1 cdc7Δ1::HIS3 Rev7-myc13 KanMX4
A364a/Pessoa-Brandão and Sclafani (2004)
A364a/Pessoa-Brandão and Sclafani (2004)
A364a/Pessoa-Brandão and Sclafani (2004)
A364a/Pessoa-Brandão and Sclafani (2004)
S288C/Minesinger and Jinks-Robertson (2005)
S288C/this study
S288C/this study
1992). Spontaneous reversion rates of the lys2DA746 frameshift were determined by the method of the median (Lea
and Coulson 1949) using data from 12–24 independent cultures. For analysis of reversion spectra, independent Lys+
revertants were isolated and an appropriate portion of the
lys2 gene was PCR amplified and sequenced (Harfe and
Jinks-Robertson 2000).
DNA damage survival and epistasis analysis
UV (254 nm) treatment of log-phase cells was as described
(Pessoa-Brandão and Sclafani 2004). Briefly, cells were exposed to defined doses of UV, plated on YPD plates, and
then surviving colonies were counted after incubation in
the dark for 2–3 days at 30°. The data presented represent
the mean and SD of three independent experiments. The
following equation describes the predicted survival of a double mutant strain if the interaction between relevant mutant
genes is additive, with S representing the surviving fraction
of cells at a given UV dose (Brendel and Haynes 1973):
2 ln Smutant 1
2ln Sdouble m
utant ¼
þ 2 ln Smutant 2 2 ð2ln SWT Þ:
If the observed 2ln Sdouble mutant is greater than predicted by
the equation, one can conclude that the interaction between
the two mutations is synergistic.
Co-immunoprecipitations and immunoblotting
Protein extracts were prepared using a glass bead lysis
procedure (Pessoa-Brandão and Sclafani 2004) and proteins
were separated by SDS–PAGE. Immunoblotting was performed with primary mouse monoclonal anti-HA (12CA5,
Roche) or anti-myc (9E10, Babco) antibodies, followed by
S288C/this study
W303/Sabbioneda et al. (2005)
A364a/Pessoa-Brandão and Sclafani (2004)
A364a/Pessoa-Brandão and Sclafani (2004)
A364a/Pessoa-Brandão and Sclafani (2004)
A364a/this study
A364a/this study
A364a/this study
A364a/this study
A364a/Oshiro et al. (1999)
A364a/this study
A364a/this study
antimouse HRP secondary antibodies or directly by using
anti-HA and anti-myc antibody HRP conjugates and subsequent visualization by enhanced chemiluminescence (Oshiro
et al. 1999; Pessoa-Brandão and Sclafani 2004; Leon et al.
2008). Anti-Pcf11 and Anti-ADH antibodies were rabbit polyclonal antibodies obtained from D. Bentley (University of
Colorado, Denver) and T. Young (University of Washington,
Seattle).
For immunoprecipitation (IP) assays, 1 mg of protein
extract was combined with 40 ml of Protein G-Sepharose 4B
Fast flow beads (Sigma-Aldrich) and 25 mg of antibody in
the presence of protease inhibitors [mini protease inhibitor
cocktail tablets (Roche Diagnostics) supplemented with 2
mg/ml pepstatin and 1 mM AEBSF (4-benzenesulfonyl fluoride hydrochloride)] in a total volume of 500 ml. Protein G
beads were blocked overnight at 4° with 3% filtered BSA in
lysis buffer (50 mM Tris-HCl pH 7.6, 50 mM NaCl, 0.1%
Triton X-100, 0.1% Tween 20, 1 mM EDTA) and then
washed three times with 1 ml of lysis buffer + protease
inhibitors before use. After overnight incubation at 4° with
end-over-end rotation, the IP reactions were pelleted at
500 3 g for 3 min. The supernatant was removed and mixed
with 53 SDS sample buffer (13 final) and immediately
boiled for 5 min. The IP pellet was washed three times with
1 ml of lysis buffer + protease inhibitors, then resuspended
in hot SDS sample buffer and boiled for 5 min. For coimmunoprecipitation (co-IP) assays, strain YSS13 containing
an endogenous Myc-tagged REV7 allele was transformed with
a high-copy plasmid encoding HA-tagged WT or kinase-dead
Cdc7 (pCH766 or pCH777, respectively) or HA-tagged Dbf4
(pYQ118). The signals on immunoblots were quantified using
Lab Works (UVP, ver. 4.5) software. In co-immunoprecipitations, we normalize the signal of the second protein by
DDK Regulates Polz and Mutagenesis
1113
Table 2 Plasmids used in this study
Plasmid
pRS426
pLPB46
pBL211
pYQ118
pCH766
pCH777
pSF2
pEG(KT)
pEG.MCM2
pJN60
pLPB60
721
Genotype
Source or reference
2m URA3 vector
2m URA3 FLAG-POL30
ARS CEN URA3 POL30
2m URA3 GAL1pro-His6-HA-DBF4
2m HIS3 GAL10pro-4xHA-CDC7
2m HIS3 GAL10pro-3xHA-cdc7(N168A)
2m URA3 Cupro-GST-REV3
2m leu2-d URA3 GAL1-10pro-GST
2m leu2-d URA3 GAL1-10pro-GST-MCM2
2m URA3 GAL1pro-GST-REV1
2m URA3 GAL1pro -GST-REV7
YIp211-P30-pol30(K127/164R)-39 URA3
Sikorski and Hieter (1989)
This study
Ayyagari et al. (1995)
Oshiro et al. (1999)
Hardy and Pautz (1996)
Hardy and Pautz (1996)
Nelson et al. (1996b)
Mitchell et al. (1993)
Lei et al. (1997)
Nelson et al. (1996a)
This study
Stelter and Ulrich (2003)
dividing by the signal of the protein immunoprecipitated. For
example, the Rev7-myc signal was divided by HA-Dbf4 signal
in an anti-HA immunoprecipitate to yield the relative amount
of Rev7-myc in the HA-Dbf4 immunoprecipitate.
For GST-pulldown assays, 1 mg of protein extract was
added to 30 ml of Glutathione Sepharose 4B beads (Amersham Pharmacia) in the presence of protease inhibitors at
a final volume of 200 ml. GST beads were washed two to
three times with 500 ml of lysis buffer + protease inhibitors
before use, and the GST-pulldown reactions were processed
in the same manner as IP reactions.
Yeast and Xenopus DDK assays
Yeast Cdc7 IP kinase assays were performed using Myctagged Cdc7 isolated from strain YJO235 and appropriate
GST-tagged target proteins as described previously (Oshiro
et al. 1999). GST-tagged fusion proteins were purified from
strain yLPB132, which contains a cdc7 KD allele (cdc7D163N) to eliminate phosphorylation of the fusion proteins
by endogenous DDK. Anti-Myc immune complexes were
washed once with kinase buffer [15 mM MgCl2, 50 mM
Tris-HCl (pH 7.6)] to remove detergents present in the lysis
buffer. To these immunoprecipitates were added 30 ml of
kinase buffer plus ATP (50 mM Tris-HCl pH 8.0, 15 mM
MgCl2, 10 mM ATP, 5mCi of g-32P ATP (6000 Ci/mmol)
and 1–5 mg of the GST-tagged substrate. Reaction mixtures
were incubated at 30° for 20 min unless otherwise indicated, and reactions were stopped by adding 10 ml of 43
SDS sample buffer and boiling for 5 min. Proteins were resolved by SDS–PAGE and transferred to nitrocellulose, and
phosphorylation was analyzed by either autoradiography or
PhosphorImager analysis (Molecular Dynamics). Assays
were linear with respect to the amount of Cdc7 immunoprecipitated in that more target phosphorylation was observed
when increasing amounts of immunoprecipitated Cdc7Myc9 were used.
Reactions using bacterially produced purified recombinant Xenopus DDK (xCdc7/xDbf4), which was obtained from
J. Walter (Harvard University, Cambridge, MA) (Takahashi
and Walter 2005), contained 10 mg DDK in 50 mM Na-HEPES
buffer pH 7.6, 10 mM MgCl2, 1 mM DTT, 10 mM ATP, 10 mCi
1114
L. N. Brandão et al.
of g-32P-ATP [6000 Ci/mmol]) and 1–5 mg of the GST substrate. Reactions were incubated at 25° for 30 min as described (Takahashi and Walter 2005) and were analyzed
similarly to the yeast Cdc7 IP reactions described above.
Results
DDK is required for both UV-induced and
spontaneous mutagenesis
Previous results demonstrated cdc7D and rev3D mutations
are epistatic with regard to survival following UV damage
(Pessoa-Brandão and Sclafani 2004), but a requirement of
DDK for UV-induced mutagenesis was not assayed. Here we
examine UV-induced forward mutations at CAN1 in the presence and absence of Cdc7 protein. Similar to rev3D mutants
(Lemontt 1971), the cdc7D mcm5-bob1 strain had an undetectable level of UV-induced CanR mutants at five different
UV doses with .300-fold reduction in mutations at the highest UV dose of 70 J/m2 (Figure 1). This is consistent with
our previous analyses of hypomorphic cdc7ts mutants, which
have reduced levels of induced mutagenesis under permissive conditions (Ostroff and Sclafani 1995).
The rate of spontaneous mutagenesis at CAN1, measured
by the level of mutation in cells without UV in Figure 1, was
reduced in the cdc7D mcm5-bob1 background, suggesting
DDK is required for spontaneous mutagenesis (1.67 for
cdc7D mcm5-bob1 vs. 9.0 for CDC7 per 106 cells). However,
CAN1 is a relatively insensitive assay as even rev3D and
rev7D strains show only small decreases in the rate of spontaneous mutagenesis. A significantly more sensitive test of
Polz relevance to spontaneous mutagenesis is achieved by
the lys2DA746 frameshift-reversion assay, where “complex”
mutations (CINS-Complex Insertions), in which the selected
frameshift is accompanied by one or more base substitution,
are a signature of Polz activity (Harfe and Jinks-Robertson
2000). This signature is most evident in the absence of nucleotide excision repair (NER), where the load of endogenous DNA damage is elevated and the need for error-prone
bypass is enhanced. We utilized the lys2DA746 reversion
assay to analyze spontaneous mutagenesis in cdc7D mcm5bob1 strains, with RAD1 additionally deleted to eliminate
Figure 1 Induced mutagenesis requires CDC7. CDC7
(RSY466) and mcm5-bob1 cdc7D (P211) congenic
strains (A364a background) were treated with different doses of UV light and plated for viability and
survival on canavanine-containing medium. Values
shown are mean of two independent experiments.
NER. The overall lys2DA746 reversion rates were similar in
rad1D mcm5-bob1, rad1D cdc7D mcm5-bob1, and rad1D
rev3D mcm5-bob1 strains (3 3 1029 in each strain). The
spectrum of revertants from the rad1D mcm5-bob1 strain
(Figure 2A) resembled the spectrum from rad1D CDC7
MCM5 strains (Harfe and Jinks-Robertson 2000), with
30% of revertants containing a complex mutation. However, the spectrum was dramatically altered in both the
rad1D cdc7D mcm5-bob1 and rad1D rev3D mcm5-bob1
strains, with an almost complete elimination of complex
mutations (Figure 2, B and C, respectively) as reported previously in rad1D rev3D strains (Harfe and Jinks-Robertson
2000). These results suggest DDK regulates both induced
and spontaneous mutagenesis catalyzed by Polz. In addition
to reduction in complex events, the rad1D cdc7D mcm5-bob1
strain also exhibited a proportional reduction in the number
of large deletions among revertants (from 12/74 in rad1D
mcm5-bob1 to 1/79). These large deletions were not affected
Figure 2 CDC7 is required for
the production of complex mutations (CINS) in the lys2DA746
frameshift reversion assay. The
spectra of mutations compiled
from independent Lys+ revertants
isolated from (A) RSY1183, (B)
RSY1190, and (C) RSY1224 are
shown. N corresponds to the
number or revertants sequenced
in each strain background.
DDK Regulates Polz and Mutagenesis
1115
by the presence or absence of Polz and have been attributed
to strand misalignment associated with a reduction in the
processivity of Pold (Minesinger and Jinks-Robertson 2005).
DDK interacts with and phosphorylates Rev7
The epistatic relationship between cdc7D and rev3D with
respect to UV survival (Pessoa-Brandão and Sclafani 2004)
and their similar affects on UV-induced and spontaneous mutagenesis (above) suggest Polz might be a target of DDK. We
thus investigated if DDK interacts with the Rev3 and Rev7
subunits of Polz. We found that a Rev7-Myc13 tagged protein
(Sabbioneda et al. 2005) was co-immunoprecipitated with
HA-tagged Dbf4, while no co-IP was seen in a mock reaction without the HA-specific antibody (Figure 3A). When
Rev7-Myc13 was immunoprecipitated, similar amounts of
HA-Cdc7 and inactive HA-Cdc7-N168A proteins (Hardy and
Pautz 1996) were co-immunoprecipitated (Figure 3B). When
HA-Cdc7 and HA-Cdc7-N168A were immunoprecipitated,
about 5–10 times more Rev7-Myc13 co-immunoprecipitated
with the inactive HA-Cdc7-N168A protein (Figure 3C), which
is consistent with observations that inactive protein kinases bind substrates more efficiently (Wu et al. 1996). Both
HA-Cdc7 and HA-Cdc7-N168A proteins appear as doublets
on these gels. The doublets have been seen previously without explanation (Hardy and Pautz 1996). It is possible it
represents phosphorylation of Cdc7 by another kinase.
No increase was observed in the co-IP of Rev7 with Cdc7
or Dbf4 following treatment of cells with 100 J/m2 UV (data
not shown). We also failed to detect any interaction of
DDK with GST-Rev3 protein using plasmid pSF2 (Table 2)
(Pessoa-Brandão 2005). Controls with a nonspecific nuclear
(Pcf11) or an abundant cytoplasmic protein (Adh1) with
either the HA-tagged Cdc7 protein or the Rev7-Myc13 protein were negative (Figure 4).
The co-IP of Rev7-Myc13 with HA-Cdc7 or HA-Dbf4 suggests that Rev7 might be a physiological target of the DDK.
We examined whether Cdc7-Myc9 immunoprecipitates,
which contain active DDK complex, can phosphorylate purified yeast GST-Rev7 protein in vitro. Yeast GST-Mcm2 was
used as the positive control for kinase activity and, as
expected, Dbf4 was also autophosphorylated in the reaction
(Figure 5A). Importantly, purified GST-Rev7 was phosphorylated when incubated with the Cdc7-Myc9 IP, while GST
alone was not. However, we estimate that GST-Rev7 is phosphorylated about two to three times less than GST-Mcm2
and may be a less efficient substrate. In support of these
findings, similar phosphorylation of GST-Rev7 was observed
using purified recombinant Xenopus DDK (Takahashi and
Walter 2005) (Figure 5B).
Rev7 protein binds to Cdc7 protein in the absence
of Dbf4
Because an inactive kinase-dead Cdc7 kinase can still bind
Rev7 protein (Figure 3), we tested whether Cdc7 kinase is
able to bind to Rev7 in the absence of Dbf4 protein (Figure
6A). Because DBF4 and CDC7 are essential genes, we used
1116
L. N. Brandão et al.
Figure 3 Co-IP of Rev7 with Dbf4 or Cdc7. Cell extracts were incubated
with anti-HA (A and C) or anti-Myc (B) antibody, and precipitated proteins
were separated by SDS–PAGE. Western blots were probed individually
with an anti-HA or anti-Myc antibody to detect tagged proteins in the
immunoprecipitate. Strain YSS13 (Rev7-Myc) with plasmid pYQ118 (A,
HA-Dbf4), pCH766 (B and C, HA-Cdc7), or pCH777 (HA-Cdc7-KD). In B
and C, extracts were from cells containing a tagged wild-type (WT) or
kinase-dead (KD) Cdc7, as indicated. Lane 1, input extract only; lane 2,
supernatant of IP; lane 3, supernatant of mock IP with no antibody; lane
4, pellet of mock IP; lane 5, pellet of IP. Twenty percent of the IP was used
for SDS–PAGE as compared to 5% of the supernatants or the input
extract.
strains containing the mcm5-bob1 mutation that bypasses
the lethality of both dbf4D and cdc7D mutations (Hardy
et al. 1997). Strain RSY1445 (Table 1), which also contains
an integrated REV7-MYC13 gene, was transformed with the
HA-tagged CDC7 plasmid pCH766 (Table 2). Even in the
absence of Dbf4 protein, Cdc7 protein was still able to coimmunoprecipitate with the Rev7-Myc13 protein (Figure
6A). Co-immunoprecipitation of Rev7-myc13 with Cdc7
appears to be less efficient when Dbf4 was absent as more
Rev7-myc13 remains in the supernatant when Dbf4 is absent (Figure 6A). However, using plasmid pYQ118 (Table 2;
HA-Dbf4) in cdc7D mcm5-bob1 strain (yLPB163; Table 1)
that contains a tagged REV7-MYC13 gene, we found the reverse is not true; Dbf4 protein cannot co-immunoprecipitate
with Rev7-Myc13 in the absence of Cdc7 protein (Figure
6B). We estimate the amount of Rev7-Myc13 protein co-
Figure 4 Control co-immunoprecipitation for nonspecific interactions
between Rev7-myc or HA-Cdc7 with Pcf11 or Adh1. Cell extracts were
directly loaded (input in lane 1) or incubated and immunoprecipitated
with anti-Myc antibody (lanes 2 and 3), anti-HA (lanes 4 and 5), or no
antibody (lines 6 and 7) and then proteins were separated by SDS–
PAGE. Western blots were probed individually with an anti-Pcf11 (top)
or anti-Adh1 antibody (bottom) to detect any tagged proteins in the
immunoprecipitate.
immunoprecipitated with HA-Dbf4 protein is at least 25- to
50-fold reduced when Cdc7 protein is absent. In contrast,
the amount of Rev7-myc13 protein co-immunoprecipitated
with HA-Cdc7 protein is only reduced 1.3-fold when Dbf4
protein is absent. We conclude Rev7 protein binding of DDK
is mediated primarily by the Cdc7 kinase subunit and does
not require the Dbf4 subunit, although Dbf4 protein likely
contributes to stabilize the binding.
Loss of DDK is not epistatic with the loss of PCNA posttranslational modifications and Pol32 in survival
following UV irradiation
We investigated whether DDK involvement in TLS was regulated by interacting with subunits of Pold that are important in Polz-dependent mutagenesis using genetic epistasis
analysis (Figure 7) (Boiteux and Jinks-Robertson 2013). Polz
also interacts with Pol32, a nonessential subunit of Pold that is
required for Pol z-dependent mutagenesis (Gerik et al. 1998;
Huang et al. 2000). In biochemical analyses, Polz holoenzyme
is a four subunit complex called Polz4 that contains Pol32
and Pol31 in addition to the core Rev3 and Rev7 proteins
(Makarova et al. 2012). Polz also interacts with PCNA
(encoded by POL30), a homotrimeric sliding clamp that promotes DNA polymerase processivity (Ayyagari et al. 1995).
PCNA is modified at K127 and K164 by sumoylation and
ubiquitination, respectively, to modulate postreplication repair
(PRR) pathways (reviewed in Boiteux and Jinks-Robertson
2013). Monoubiquitination of PCNA by Rad6-Rad18 promotes
TLS by Polz and Polh; the pol30-K164R and pol30-K127R,
K164R alleles are epistatic with rev3D for survival following
UV irradiation (Stelter and Ulrich 2003). Polyubiquitination of
monoubiqutinated PCNA by Ubc13-Mms2-Rad5 promotes
error-free damage avoidance (Hoege et al. 2002).
Given the epistasis between cdc7D and rev3D with respect
to survival following UV treatment, we examined whether
the pol30-K127R,K164R or pol32D allele is similarly epistatic
to a cdc7 null mutation. Instead of epistasis, we observed at
Figure 5 Phosphorylation of yeast GST-Rev7 by yeast and Xenopus DDK.
A total of 2–4 mg of the indicated GST-tagged yeast protein were incubated with (A) Cdc7-Myc immunopreciptates from strain YJO235 or (B)
10 mg of Xenopus DDK (xCdc7-xDbf4) in the presence of g32P-ATP.
Reactions were subjected to SDS–PAGE, proteins were transferred to
nitrocellulose, and protein phosphorylation was visualized by autoradiography. Lanes in A were from different gels and aligned by the molecular
weight standards. Anti-GST (UBI) blots of the filters showed the position
and confirmed equivalent amounts of the indicated GST fusion proteins
(not shown). (A) Lane 1, Cdc7-Myc IP plus GST-Mcm2 (103 kDA); lane 2,
Cdc7-Myc IP plus GST-Rev7 (55 kDA); lane 3, Cdc7-Myc IP plus GST (22
kDA); lane 4, Cdc7-Myc only. Note autophosphorylation of Dbf4 by the
Cdc7-Myc IP in lanes 2–4. (B) Lane 1, Xenopus DDK only; lane 2, GST
only; lane 3, GST-Mcm2 only; lane 4, GST-Rev7 only; lane 5, GST plus
DDK; lane 6, GST-Mcm2 plus DDK; lane 7, GST-Rev7 plus DDK. Note DDK
autophosphorylation of both xDbf4 and xCdc7 subunits.
least an additive and possible synergistic interaction between pol30-K127R,K164R and cdc7D mcm5-bob1 at three
different UV doses (Figure 7A, Table 3). Furthermore,
pol32D also showed a nonepistatic relationship with cdc7D
mcm5-bob1 (Figure 7B, Table 3). We also examined possible
physical interactions of Cdc7 and Dbf4 with GST-Rev1,
Pol32-HA, or FLAG-Pol30 proteins using co-IP and GST
pulldown experiments but failed to detect any interactions
(Pessoa-Brandão 2005).
Discussion
In this report, we have investigated the role of DDK in Polzdependent TLS and mutagenesis. Previous studies showed
DDK regulates mutagenesis induced by various DNA
damaging agents (Figure 1) (Njagi and Kilbey 1982;
Hollingsworth et al. 1992; Ostroff and Sclafani 1995; Pessoa-Brandão and Sclafani 2004), but DDK had not been
similarly implicated in spontaneous mutagenesis (Sclafani
2000). Because DDK and Polz are in the same DNA-
DDK Regulates Polz and Mutagenesis
1117
Figure 6 Analysis of the co-IP of Rev7 by each subunit of DDK. Cell
extracts were incubated with anti-HA antibody, and precipitated proteins
were separated by SDS–PAGE. Western blots were probed individually
with an anti-HA or anti-Myc antibody to detect tagged proteins in the
immunoprecipitate. (A) WT strain YSS13 (lanes 1–5) or dbf4D strain
RSY1445 (lanes 6–10) with HA-Cdc7 plasmid pCH766 was used. (B)
WT strain YSS13 (lanes 1–5) or cdc7D strain yLPB163 (lanes 6–10) with
HA-Dbf4 plasmid pYQ118 was used. Lanes are labeled the same in A and
B. Lane 1, input extract only; lane 2, supernatant of mock IP with no
antibody; lane 3, supernatant of IP; lane 4, pellet of mock IP; lane 5, pellet
of IP; lane 6, input extract only; lane 7, supernatant of mock IP with no
antibody; lane 8, supernatant of IP; lane 9, pellet of mock IP; lane 10,
pellet of IP; 20% of the IP was used for SDS–PAGE as compared to 5% of
the supernatants or the input extract.
damage response pathway (Pessoa-Brandão and Sclafani
2004) and Polz is required for spontaneous mutagenesis,
the role of DDK in spontaneous mutagenesis was investigated using the sensitive lys2DA746 reversion assay (Harfe
and Jinks-Robertson 2000). Significantly, we found DDK is
required for the production of complex frameshift events
(CINS), which are a distinctive mutational signature of Polz
(Harfe and Jinks-Robertson 2000; Sabbioneda et al. 2005)
(Figure 2). We conclude DDK is required for Polz-dependent
spontaneous, as well as induced, mutagenesis.
In addition to a reduction in CINS, the cdc7D mcm5bob1 strain also showed a reduction in the number of large
deletions that revert the lys2DA746 allele (Figure 2).
These large deletions have been attributed to a decrease
in Pold processivity, as they increase in pol30-46 and
pol32D mutants (Minesinger and Jinks-Robertson 2005).
The large deletions are thought to be produced when
1118
L. N. Brandão et al.
lesion-stalled replication restarts downstream (39) during
lagging-strand synthesis. It has been suggested that DDK
may be required to facilitate replication restart after
checkpoint activation (Duncker and Brown 2003), and
this, in principle, could explain the loss of large deletions
in the cdc7D mcm5-bob1 background. From our recent
data, we have ruled out this hypothesis because replication restart not only occurs in cdc7D mcm5-bob1 strains,
but the restarted replication forks proceed at a faster rate
than in wild-type or mcm5-bob1 cells (Zhong et al. 2013).
Presumably, the increased fork rate reflects an excess of
replication factors and reduced Rad53 checkpoint signaling due to reduced origin firing. Based on the previously
discussed processivity hypothesis (Minesinger and JinksRobertson 2005), we propose the reduction in large deletions in the cdc7D mcm5-bob1 mutant may result from
increased processivity when a replication fork restarts after encountering DNA damage.
The role of DDK in the DNA damage checkpoint is
equivocal in that it is not clear whether DDK activity itself
or some downstream event is inhibited during DNA damage
(Sclafani and Holzen 2007). Yeast DDK function is inhibited
and late origin firing is blocked by Rad53 phosphorylation
of the DDK regulatory subunit Dbf4 (Chen et al. 2013); mutation of the Dbf4 phosphorylation sites to alanines removes
the inhibition (Zegerman and Diffley 2009; Lopez-Mosqueda
et al. 2010). However, even though MCM proteins are known
DDK substrates during replication (Sclafani and Holzen
2007), DDK phosphorylation of Mcm2 protein in vitro is not
reduced (Oshiro et al. 1999) and phosphorylation of the
Mcm7 protein is only modestly reduced during checkpoint
activation (Weinreich and Stillman 1999). Furthermore,
yeast Mcm4 is still phosphorylated during DNA replication-checkpoint arrest in vivo (Sheu and Stillman 2006).
Recently, it was shown that DDK phosphorylation of yeast
Eco1, which is required to establish chromosome cohesion,
is inhibited by Rad53 phosphorylation of Dbf4 (Lyons et al.
2013). Similarly, DDK phosphorylation of Mer2 during
meiosis is inhibited by Rad53 phosphorylation of Dbf4
(Blitzblau and Hochwagen 2013). Yet, in human cells,
DDK phosphorylates both Claspin (Kim et al. 2008) and
hMcm2 (Tenca et al. 2007) during DNA damage both
in vivo and in vitro. Therefore, DDK is active during checkpoint responses depending on the system, substrate, and
assay used. Based on the current report, we infer that
DDK must be active during DNA damage checkpoint activation to allow for TLS. Similarly, human DDK on chromatin
remains active during checkpoint activation to facilitate
TLS by Polh (Yamada et al. 2013). It was proposed there
are two distinct pools of DDK with the soluble pool
inhibited to block late origin firing, while the chromatinbound pool remains active to facilitate TLS (Yamada et al.
2013). It is also possible the effect of checkpoint activation
on DDK function is either substrate specific (Blitzblau and
Hochwagen 2013; Lyons et al. 2013) or affects different
downstream effectors.
Figure 7 Survival of single- and double-mutant
strains following UV irradiation. (A) UV sensitivity
of cdc7Δ mcm5-bob1 with pol30-K127,K164R. (B)
UV sensitivity of cdc7Δ mcm5-bob1 with pol32D.
Viability of relevant mutant strains was determined
following exposure of log-phase cultures to UV.
Error bars represent the standard deviation of
the mean value of three independent experiments.
As an example of DDK being active during checkpoint
activation, human Rad18 is phosphorylated by DDK to
produce a binding site for Polh during TLS at sites of
replication fork stalling (Day et al. 2010; Yamada et al.
2013). However, the “S box” region of human Rad18 that
is phosphorylated by human DDK is not conserved in
either budding or fission yeast and it was suggested
DDK in yeast may phosphorylate other TLS proteins
(Day et al. 2010). In support of this idea, we show in this
report that Rev7 subunit of Polz may be the target as DDK
binds to (Figure 3) and phosphorylates Rev7 protein
(Figure 5). Therefore, there is conservation of DDK function as DDK regulates TLS polymerases in both humans
and yeast.
Furthermore, we show Rev7 binds largely to the Cdc7
kinase subunit and not to the Dbf4 regulatory subunit (Figure 6). Although Dbf4 protein is required for Cdc7 kinase to
phosphorylate substrates (Kitamura et al. 2011), and may
bind substrates in the Zn++ binding domain of motif C
(Hughes et al. 2012), it has not been determined whether
Cdc7 or Dbf4 protein alone can bind substrate proteins. In
contrast, the N-terminal domain of Dbf4 is required for DDK
to bind to MCM2–7 complex in the pre-replication complex
in vivo (Duncker et al. 2002; Varrin et al. 2005) and in vitro
(Sheu and Stillman 2006; Francis et al. 2009). In cyclindependent kinases (CDKs), the main recognition site for the
substrate is in the CDK subunit, while the cyclin hydrophobic patch stabilizes the interaction and increases potential
DDK Regulates Polz and Mutagenesis
1119
Table 3 Mathematical analysis of UV survival data
Strain–genotype
yLPB226 cdc7D pol30-K127,
K164R
yLPB95 cdc7D pol32D
a
UV dose (J/m2)
Expected 2ln Sdma
(for additive interaction)
Observed 2ln Sdma
20
2.29
3.16 6 0.24
50
70
20
50
70
4.42
6.43
1.46
2.27
3.91
5.52
6.91
1.63
3.27
4.96
0.05
0.11
0.10
0.12
0.13
Sdm, surviving fraction for double mutant. If the 2ln Sdm observed is greater than expected, then the interaction is synergistic
(Brendel and Haynes 1973).
for substrate phosphorylation (reviewed in Morgan 2007). By
analogy, we propose Cdc7 protein can bind Rev7 without Dbf4
unlike binding to the Mcm4 substrate, which requires Dbf4.
Although there are a number of possible interpretations
of additive and synergistic interactions with DNA repair
pathways (Brendel and Haynes 1973), one interpretation of
the interaction seen between cdc7D and pol32D or pol30K127R,K164R mutations (Figure 7, Table 3) is Pol32 protein
and the Pol30 (PCNA) modifications regulate multiple TLS
and also replicative polymerases (Boiteux and Jinks-Robertson
2013), while DDK regulates TLS differently by regulating Polz
through regulation of Rev7. DDK may regulate TLS lesion
bypass during “gap-filling” rather than during “polymerase
switching” (Waters et al. 2009). In the gap-filling model, repriming of replication occurs downstream of the lesion leaving
a gap, which is subsequently filled in by TLS. This is consistent
with the pol30 mutant being more sensitive to UV than the
cdc7 mutant (Figure 7).
Previously, we have shown Cdc7 kinase activity to be required for all three DDK functions: DNA replication, meiosis,
and mutagenesis as a Cdc7 KD mutant is null in all three
cellular systems (reviewed in Sclafani and Jackson 1994).
Similar to DDK’s role in DNA replication in which phosphorylation of the MCM helicase loads Cdc45 and GINS proteins
to initiate replication (Tanaka and Araki 2013), and in meiosis, where DDK phosphorylation loads Mer2 onto chromatin to initiate recombination (Matos et al. 2008; Wan et al.
2008), we propose DDK is likely to regulate Polz by phosphorylating Rev7 and loading it onto chromatin. Once on
chromatin, Rev7 would then bind to Rev3 polymerase subunit to produce an active Polz complex. This may explain
why we were unable to coIP Rev3 with DDK as Rev3-Rev7DDK complexes may not exist in the cell. Consistent with
this idea, human DDK phosphorylates Rad18 on chromatin
to load Polh for TLS (Day et al. 2010; Yamada et al. 2013).
Thus, we speculate DDK has evolved to regulate TLS and
mutagenesis (Yamada et al. 2013) and to act as a multipurpose regulator for loading of proteins onto substrate DNA
and chromatin.
Acknowledgments
Cassidy Punt, a student at the University of Colorado Master
of Science program, performed some yeast and plasmid
1120
6
6
6
6
6
L. N. Brandão et al.
strain constructions. Marianne Tecklenburg provided excellent technical assistance. We thank Helle Ulrich (Max Planck
Institute), Chris Hardy (Vanderbilt University) and Marco
Muzi-Falconi (University of Milan) for strains and plasmids.
This work was supported by Public Health Service awards to
R.A.S. (R01 GM35078) and to S.J.-R. (R01 GM64769),
a National Research Service Award fellowship to R.F.
(F32GM100644) and an Institutional Research and Career
Development Award to I.S. (K12 GM000680). L.N.B. was
supported by a National Institutes of Health training grant
(T32 GM008730).
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Communicating editor: J. Nickoloff