The Role of Dbf4-Dependent Protein Kinase in DNA Polymerase Ζ

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, †Department of Biology, Emory University, Atlanta, Georgia 30322, and ‡Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710

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.TheRev7 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 Polz- dependent 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)andPol32.

NOWLEDGE of how mutations are produced is impor- block the replicative DNA polymerases (Pols a, d, and e) Ktant for understanding genetic variability in populations during S phase and require bypass by an alternative error- and the process of evolution. Cells have evolved sophisti- free or error-prone mechanism. Error-free bypass usually cated molecular mechanisms for maintaining the integrity involves a template switch to the undamaged sister chroma- of the genome. Most DNA repair mechanisms have high tid, while error-prone bypass uses translesion synthesis ﬁdelity and prevent mutations by removing damaged DNA (TLS) DNA polymerases to synthesize new DNA directly and replacing the resulting gap using the undamaged, com- across the lesion. There are at least 15 identiﬁed TLS poly- plementary strand as a template (Waters et al. 2009; Boiteux merases in human cells but only three in the yeast Saccha- and Jinks-Robertson 2013). If not repaired, some lesions romyces cerevisiae: Polz, Polh, and Rev1, which are encoded by the REV3-REV7, RAD30, and REV1 genes, respectively

Copyright © 2014 by the Genetics Society of America (Boiteux and Jinks-Robertson 2013). TLS polymerases have doi: 10.1534/genetics.114.165308 low ﬁdelity and processivity on undamaged DNA templates Manuscript received April 14, 2014; accepted for publication May 23, 2014; published and lack an associated exonuclease proofreading activity. Early Online May 28, 2014. 1These authors contributed equally to this work. The misregulation or loss of TLS polymerases is known to 2Present address: Gevo, 345 Inverness Dr. S., Bldg. C, Ste. 310, Englewood, CO result in a number of human diseases, underscoring the 80112. 3Present address: Department of Biology, 7300 Reinhardt Circle, Reinhardt importance of this process. Patients suffering from a variant University, Waleska, GA 30183. form of xeroderma pigmentosum, for example, lack Polh 4 Corresponding author: Department of Biochemistry and Molecular Genetics, and have an increased risk forskincancer(Kennedyand University of Colorado, 12801 E. 17th Ave, MS8101, Aurora, CO 80045. E-mail: [email protected] 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 were induced by the addition galactose to 2% for 2 hr. Because to recruit Rev1, a nontemplate directed deoxycytidyl ter- pSF2 has a copper-inducible promoter, pSF2-containing cells minal transferase, to the site of DNA damage (Kim et al. were induced by the addition of 0.1 mM CuSO4 for 2 hr. 2012). Genetic methods for yeast strain construction, tetrad In early studies by Kilbey (Njagi and Kilbey 1982) and analysis, and transformation were as previously described subsequent studies by our laboratory, induced mutagenesis in S. (Pessoa-Brandão and Sclafani 2004). Gene deletions marked cerevisiae was reduced in hypomorphic cdc7 mutants including with the kanMX cassette were constructed by PCR amplifica- an inactive cdc7 kinase-dead (KD) mutant (Hollingsworth et al. tion of an appropriate disruption cassette, transformation, 1992; Ostroff and Sclafani 1995; Pessoa-Brandão and Sclafani and subsequent selection with G418 (Winzeler et al. 1999). 2004). Dbf4-dependent kinase (DDK) is conserved from yeast The pol30-K127,K164R allele was introduced at the POL30 to humans and is composed of the Cdc7 kinase catalytic and locus by two-step allele replacement using XbaI-digested plas- Dbf4 regulatory subunit. Dbf4 is low in G1 phase of the cell mid 721 and verified by sequencing genomic DNA. The cycle and required for kinase activity (Sclafani and Holzen mcm5-bob1-2 allele contains a CT-to-TC mutation that con- 2007). DDK is a Ser/Thr protein kinase that regulates the verts codon 83 from proline to leucine and creates a DdeI site. initiation of DNA replication by phosphorylating one or This allele was introduced into strain SJR1177 by two-step more members of the Mcm2–7 DNA helicase (Sheu and allele replacement using MluI-digested pRAS490, which con- Stillman 2006; Randell et al. 2010; Sheu and Stillman tains mcm5-bob1-2 in pRS306 (Pessoa-Brandão and Sclafani 2010; Heller et al. 2011; reviewed in Tanaka and Araki 2004; Dohrmann and Sclafani 2006). The cdc7D::HIS3 allele 2013). Either mcm5 (mcm5-bob1) (Hardy et al. 1997) or was introduced into RSY1183 by one-step allele replacement mcm4 (mcm4D74-174) (Sheu and Stillman 2010) mutations using a 3-kb MluI–EcoRI fragment from pRS277-cdc7D::HIS3 can bypass the requirement for DDK during replication. (Jackson et al. 1993). Presence of the cdc7D::HIS3 allele was However, cdc7D mcm5-bob1, dbf4D mcm5-bob1,orcdc7D verified by restriction digest and by loss of cdc7 genetic com- dbf4D mcm5-bob1 strains are defective in other DDK func- plementation. Strain YSS13 contains Myc-tagged Rev7 and tions, including the regulation of meiotic recombination was obtained from Marco Muzi-Falconi (University of Milan, (Matos et al. 2008; Wan et al. 2008) and resistance to DNA Milan, Italy) (Sabbioneda et al. 2005). damaging agents such as UV and MMS (Pessoa-Brandão and pLPB60 encodes GST-tagged Rev7 and was constructed Sclafani 2004), suggesting the kinase interacts with and by insertion of REV7 into the NcoI/BamHI sites of the phosphorylates additional substrates that promote mutagen- pYES263 expression vector (Melcher 2000). Plasmid pLPB46, esis and meiotic recombination (Sclafani 2000). In this re- which inserts a FLAG (Sigma-Aldrich) epitope tag immedi- port, we investigated the role of DDK in mutagenesis and ately after the initiating methionine of Pol30, was con- demonstrate DDK is required for spontaneous as well as in- structed by PCR-overlap mutagenesis (Ho et al. 1989). duced Polz-dependent mutagenesis. Based on biochemical Plasmid pBL211 was used as template for PCR using out- analyses, we suggest DDK regulates mutagenesis by direct in- side primers M13Fwd (59-TGTAAAACGACGGCCAGT-39) teraction with and phosphorylation of the Rev7 subunit of and M13Rev (59-TCACACAGGAAACAGCTATGAC-39), com- Polz. Our results are important, given the conservation of plementary to the plasmid backbone, and internal primers DDK’s role in TLS in human cells (Day et al. 2010; Yamada Pol30-FlagFwd (59-ATGgattacaaggatgacgacgataagTTAGAA et al. 2013), and are significant with regard to human disease GCAAAATTTGAAGAAGC-39) and Pol30-FlagRev (59-cttatcg because overexpression of yeast DDK increases mutagenesis tcgtcatccttgtaatcCATTTTTTCTCTCTTTTGACTGCG-39;lowercase (Sclafani et al. 1988; Sclafani and Jackson 1994), human letters indicate the FLAG epitope tag). The resulting PCR DDK overexpression is observed in many types of cancer cells fragment, which includes the FLAG-Pol30 construct and (Hess et al. 1998; Nambiar et al. 2007; Bonte et al. 2008; the POL30 promoter, was then digested with XhoI and Clarke et al. 2009; Kulkarni et al. 2009), and DDK is a thera- BamHI and cloned into pRS426. The FLAG-Pol30 gene peutic target in cancer patients (Rodriguez-Acebes et al. 2010). was functional as plasmid pLPB46 complemented a lethal pol30D::kanMX4 allele. All tagged constructs used in this study (Table 1 and Table Materials and Methods 2) have been shown to function normally by genetic complementation: Cdc7-myc9 (Oshiro et al. 1999), HA-Dbf4 (Oshiro Yeast strains, media, and plasmids et al. 1999), HA-Cdc7 (Hardy and Pautz 1996), GST-Mcm2 Yeast strains and plasmids used are listed in Table 1 and (Lei et al. 1997), GST-Rev1 and GST-Rev3 (Nelson et al. Table 2, respectively. Yeast strains were grown in yeast extract/ 1996), and REV7-myc13 (Sabbioneda et al. 2005). GST-Rev7 peptone/dextrose (YPD) with 2% glucose or in synthetic produced in this study was tested by complementation of defined minimal media supplemented with appropriate amino a rev7D::KanMX4 null mutant. acids and 2% glucose (Pessoa-Brandão and Sclafani 2004). Mutagenesis assays Galactose induction of tagged genes for subsequent immunoprecipitation was as described (Oshiro et al. 1999). Briefly, UV mutagenesis assays using the CAN1 forward mutation exponentially growing cells using raffinose as carbon source system were as described previously (Hollingsworth et al.

1112 L. N. Brandão et al. Table 1 S. cerevisiae strains used in this study

Strains Relevant genotype Background/source or reference RSY299 MATa his3Δ1 leu2 trp1 ura3 can1 cyh2 A364a/Pessoa-Brandão and Sclafani (2004) RSY466 MATa leu2 ura3 trp1 his7 A364a/Pessoa-Brandão and Sclafani (2004) P211 MATa ura3 lys2 cyh2 his3 leu2 cdc7Δ::HIS3 mcm5-bob1-1 A364a/Pessoa-Brandão and Sclafani (2004) P235 MATa ura3 lys2 cyh2 his3 leu2 cdc7Δ::HIS3 dbf4Δ::URA3 mcm5-bob1-1 A364a/Pessoa-Brandão and Sclafani (2004) SJR1177 MATa ade2-101oc his3D200 ura3DNco lys2DA746 rad1::hisG S288C/Minesinger and Jinks-Robertson (2005) RSY1183 MATa ade2-101oc his3D200 ura3DNco lys2DA746 rad1::hisG mcm5-bob1-2 S288C/this study RSY1190 MATa ade2-101oc his3D200 ura3DNco lys2DA746 rad1::hisG mcm5-bob1-2 S288C/this study cdc7D::HIS3 RSY1224 MATa ade2-101oc his3D200 ura3DNco lys2DA746 rad1::hisG mcm5-bob1-2 S288C/this study rev3D::kanMX4 YSS13 MATa ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 RAD5 REV7-myc13:: W303/Sabbioneda et al. (2005) kanMX4 yLPB21 MATa ura3 his3 leu2 cyh2 trp1 can1 A364a/Pessoa-Brandão and Sclafani (2004) yLPB24 MATa ura3 his3 leu2 cyh2 cdc7Δ::HIS3 mcm5-bob1-1 A364a/Pessoa-Brandão and Sclafani (2004) yLPB91 MATa ura3 his3 leu2 cyh2 trp1 can1 pol32Δ::kanMX4 A364a/Pessoa-Brandão and Sclafani (2004) yLPB95 MATa ura3 his3 leu2 cyh2 lys2 cdc7Δ::HIS3 mcm5-bob1-1 pol32Δ::kanMX4 A364a/this study yLPB224 MATa his3 leu2 trp1 ura3 can1 cyh2 pol30-K127R,K164R A364a/this study yLPB226 MATa ura3 lys2 cyh2 his3 leu2 cdc7Δ1::HIS3+ mcm5-bob1-1 pol30-K127R,K164R A364a/this study yLPB132 MATa ura3 leu2 trp1 his7 mcm5-bob1 cdc7(D163N)-myc9 A364a/this study YJO235 MATa ura3 leu2 trp1 bar1::LEU2 CDC7-myc9 A364a/Oshiro et al. (1999) RSY1445 MATa ura3 leu2 his3 mcm5-bob1 cdc7Δ1::LEU2 dbf4D::URA3 Rev7-myc13 KanMX4 A364a/this study yLPB163 MATa ura3 leu2 his3 mcm5-bob1 cdc7Δ1::HIS3 Rev7-myc13 KanMX4 A364a/this study

1992). Spontaneous reversion rates of the lys2DA746 frame- antimouse HRP secondary antibodies or directly by using shift were determined by the method of the median (Lea anti-HA and anti-myc antibody HRP conjugates and subse- and Coulson 1949) using data from 12–24 independent cul- quent visualization by enhanced chemiluminescence (Oshiro tures. For analysis of reversion spectra, independent Lys+ et al. 1999; Pessoa-Brandão and Sclafani 2004; Leon et al. revertants were isolated and an appropriate portion of the 2008). Anti-Pcf11 and Anti-ADH antibodies were rabbit poly- lys2 gene was PCR amplified and sequenced (Harfe and clonal antibodies obtained from D. Bentley (University of Jinks-Robertson 2000). Colorado, Denver) and T. Young (University of Washington, Seattle). DNA damage survival and epistasis analysis For immunoprecipitation (IP) assays, 1 mg of protein UV (254 nm) treatment of log-phase cells was as described extract was combined with 40 ml of Protein G-Sepharose 4B (Pessoa-Brandão and Sclafani 2004). Briefly, cells were ex- Fast flow beads (Sigma-Aldrich) and 25 mg of antibody in posed to defined doses of UV, plated on YPD plates, and the presence of protease inhibitors [mini protease inhibitor then surviving colonies were counted after incubation in cocktail tablets (Roche Diagnostics) supplemented with 2 the dark for 2–3 days at 30°. The data presented represent mg/ml pepstatin and 1 mM AEBSF (4-benzenesulfonyl fluo- the mean and SD of three independent experiments. The ride hydrochloride)] in a total volume of 500 ml. Protein G following equation describes the predicted survival of a dou- beads were blocked overnight at 4° with 3% filtered BSA in ble mutant strain if the interaction between relevant mutant lysis buffer (50 mM Tris-HCl pH 7.6, 50 mM NaCl, 0.1% genes is additive, with S representing the surviving fraction Triton X-100, 0.1% Tween 20, 1 mM EDTA) and then of cells at a given UV dose (Brendel and Haynes 1973): washed three times with 1 ml of lysis buffer + protease inhibitors before use. After overnight incubation at 4° with 2 ¼ 2 ln Sdouble mutant ln Smutant 1 end-over-end rotation, the IP reactions were pelleted at þ 2 ln Smutant 2 2 ð2ln SWTÞ: 500 3 g for 3 min. The supernatant was removed and mixed with 53 SDS sample buffer (13 final) and immediately If the observed 2ln Sdouble mutant is greater than predicted by boiled for 5 min. The IP pellet was washed three times with the equation, one can conclude that the interaction between 1 ml of lysis buffer + protease inhibitors, then resuspended the two mutations is synergistic. inhotSDSsamplebufferandboiledfor5min.Forco- immunoprecipitation (co-IP) assays, strain YSS13 containing Co-immunoprecipitations and immunoblotting an endogenous Myc-tagged REV7 allele was transformed with Protein extracts were prepared using a glass bead lysis a high-copy plasmid encoding HA-tagged WT or kinase-dead procedure (Pessoa-Brandão and Sclafani 2004) and proteins Cdc7 (pCH766 or pCH777, respectively) or HA-tagged Dbf4 were separated by SDS–PAGE. Immunoblotting was per- (pYQ118). The signals on immunoblots were quantified using formed with primary mouse monoclonal anti-HA (12CA5, Lab Works (UVP, ver. 4.5) software. In co-immunoprecipita- Roche) or anti-myc (9E10, Babco) antibodies, followed by tions, we normalize the signal of the second protein by

DDK Regulates Polz and Mutagenesis 1113 Table 2 Plasmids used in this study

Plasmid Genotype Source or reference pRS426 2m URA3 vector Sikorski and Hieter (1989) pLPB46 2m URA3 FLAG-POL30 This study pBL211 ARS CEN URA3 POL30 Ayyagari et al. (1995) pYQ118 2m URA3 GAL1pro-His6-HA-DBF4 Oshiro et al. (1999) pCH766 2m HIS3 GAL10pro-4xHA-CDC7 Hardy and Pautz (1996) pCH777 2m HIS3 GAL10pro-3xHA-cdc7(N168A) Hardy and Pautz (1996) pSF2 2m URA3 Cupro-GST-REV3 Nelson et al. (1996b) pEG(KT) 2m leu2-d URA3 GAL1-10pro-GST Mitchell et al. (1993) pEG.MCM2 2m leu2-d URA3 GAL1-10pro-GST-MCM2 Lei et al. (1997) pJN60 2m URA3 GAL1pro-GST-REV1 Nelson et al. (1996a) pLPB60 2m URA3 GAL1pro -GST-REV7 This study 721 YIp211-P30-pol30(K127/164R)-39 URA3 Stelter and Ulrich (2003) dividing by the signal of the protein immunoprecipitated. For of g-32P-ATP [6000 Ci/mmol]) and 1–5 mg of the GST sub- example, the Rev7-myc signal was divided by HA-Dbf4 signal strate. Reactions were incubated at 25° for 30 min as de- in an anti-HA immunoprecipitate to yield the relative amount scribed (Takahashi and Walter 2005) and were analyzed of Rev7-myc in the HA-Dbf4 immunoprecipitate. similarly to the yeast Cdc7 IP reactions described above. For GST-pulldown assays, 1 mg of protein extract was added to 30 ml of Glutathione Sepharose 4B beads (Amer- Results sham Pharmacia) in the presence of protease inhibitors at a final volume of 200 ml. GST beads were washed two to DDK is required for both UV-induced and three times with 500 ml of lysis buffer + protease inhibitors spontaneous mutagenesis before use, and the GST-pulldown reactions were processed Previous results demonstrated cdc7D and rev3D mutations in the same manner as IP reactions. are epistatic with regard to survival following UV damage (Pessoa-Brandão and Sclafani 2004), but a requirement of Yeast and Xenopus DDK assays DDK for UV-induced mutagenesis was not assayed. Here we Yeast Cdc7 IP kinase assays were performed using Myc- examine UV-induced forward mutations at CAN1 in the pres- tagged Cdc7 isolated from strain YJO235 and appropriate ence and absence of Cdc7 protein. Similar to rev3D mutants GST-tagged target proteins as described previously (Oshiro (Lemontt 1971), the cdc7D mcm5-bob1 strain had an unde- et al. 1999). GST-tagged fusion proteins were purified from tectable level of UV-induced CanR mutants at five different strain yLPB132, which contains a cdc7 KD allele (cdc7- UV doses with .300-fold reduction in mutations at the high- D163N) to eliminate phosphorylation of the fusion proteins est UV dose of 70 J/m2 (Figure 1). This is consistent with by endogenous DDK. Anti-Myc immune complexes were our previous analyses of hypomorphic cdc7ts mutants, which washed once with kinase buffer [15 mM MgCl2,50mM have reduced levels of induced mutagenesis under permis- Tris-HCl (pH 7.6)] to remove detergents present in the lysis sive conditions (Ostroff and Sclafani 1995). buffer. To these immunoprecipitates were added 30 mlof The rate of spontaneous mutagenesis at CAN1, measured kinase buffer plus ATP (50 mM Tris-HCl pH 8.0, 15 mM by the level of mutation in cells without UV in Figure 1, was 32 MgCl2,10mM ATP, 5mCi of g- P ATP (6000 Ci/mmol) reduced in the cdc7D mcm5-bob1 background, suggesting and 1–5 mg of the GST-tagged substrate. Reaction mixtures DDK is required for spontaneous mutagenesis (1.67 for were incubated at 30° for 20 min unless otherwise indi- cdc7D mcm5-bob1 vs. 9.0 for CDC7 per 106 cells). However, cated, and reactions were stopped by adding 10 mlof43 CAN1 is a relatively insensitive assay as even rev3D and SDS sample buffer and boiling for 5 min. Proteins were re- rev7D strains show only small decreases in the rate of spon- solved by SDS–PAGE and transferred to nitrocellulose, and taneous mutagenesis. A significantly more sensitive test of phosphorylation was analyzed by either autoradiography or Polz relevance to spontaneous mutagenesis is achieved by PhosphorImager analysis (Molecular Dynamics). Assays the lys2DA746 frameshift-reversion assay, where “complex” were linear with respect to the amount of Cdc7 immunopre- mutations (CINS-Complex Insertions), in which the selected cipitated in that more target phosphorylation was observed frameshift is accompanied by one or more base substitution, when increasing amounts of immunoprecipitated Cdc7- are a signature of Polz activity (Harfe and Jinks-Robertson Myc9 were used. 2000). This signature is most evident in the absence of nu- Reactions using bacterially produced purified recombi- cleotide excision repair (NER), where the load of endoge- nant Xenopus DDK (xCdc7/xDbf4), which was obtained from nous DNA damage is elevated and the need for error-prone J. Walter (Harvard University, Cambridge, MA) (Takahashi bypass is enhanced. We utilized the lys2DA746 reversion and Walter 2005), contained 10 mg DDK in 50 mM Na-HEPES assay to analyze spontaneous mutagenesis in cdc7D mcm5- buffer pH 7.6, 10 mM MgCl2,1mMDTT,10mMATP,10mCi bob1 strains, with RAD1 additionally deleted to eliminate

1114 L. N. Brandão et al. 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 strains, with an almost complete elimination of complex rad1D mcm5-bob1, rad1D cdc7D mcm5-bob1, and rad1D mutations (Figure 2, B and C, respectively) as reported pre- rev3D mcm5-bob1 strains (3 3 1029 in each strain). The viously in rad1D rev3D strains (Harfe and Jinks-Robertson spectrum of revertants from the rad1D mcm5-bob1 strain 2000). These results suggest DDK regulates both induced (Figure 2A) resembled the spectrum from rad1D CDC7 and spontaneous mutagenesis catalyzed by Polz. In addition MCM5 strains (Harfe and Jinks-Robertson 2000), with to reduction in complex events, the rad1D cdc7D mcm5-bob1 30% of revertants containing a complex mutation. How- strain also exhibited a proportional reduction in the number ever, the spectrum was dramatically altered in both the of large deletions among revertants (from 12/74 in rad1D rad1D cdc7D mcm5-bob1 and rad1D rev3D mcm5-bob1 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 Figure 3 Co-IP of Rev7 with Dbf4 or Cdc7. Cell extracts were incubated DDK with GST-Rev3 protein using plasmid pSF2 (Table 2) with anti-HA (A and C) or anti-Myc (B) antibody, and precipitated proteins (Pessoa-Brandão 2005). Controls with a nonspecific nuclear were separated by SDS–PAGE. Western blots were probed individually (Pcf11) or an abundant cytoplasmic protein (Adh1) with with an anti-HA or anti-Myc antibody to detect tagged proteins in the immunoprecipitate. Strain YSS13 (Rev7-Myc) with plasmid pYQ118 (A, either the HA-tagged Cdc7 protein or the Rev7-Myc13 pro- HA-Dbf4), pCH766 (B and C, HA-Cdc7), or pCH777 (HA-Cdc7-KD). In B tein were negative (Figure 4). and C, extracts were from cells containing a tagged wild-type (WT) or The co-IP of Rev7-Myc13 with HA-Cdc7 or HA-Dbf4 sug- kinase-dead (KD) Cdc7, as indicated. Lane 1, input extract only; lane 2, gests that Rev7 might be a physiological target of the DDK. supernatant of IP; lane 3, supernatant of mock IP with no antibody; lane We examined whether Cdc7-Myc9 immunoprecipitates, 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 which contain active DDK complex, can phosphorylate puri- extract. fied 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 strains containing the mcm5-bob1 mutation that bypasses (Figure 5A). Importantly, purified GST-Rev7 was phosphor- the lethality of both dbf4D and cdc7D mutations (Hardy ylated when incubated with the Cdc7-Myc9 IP, while GST et al. 1997). Strain RSY1445 (Table 1), which also contains alone was not. However, we estimate that GST-Rev7 is phos- an integrated REV7-MYC13 gene, was transformed with the phorylated about two to three times less than GST-Mcm2 HA-tagged CDC7 plasmid pCH766 (Table 2). Even in the and may be a less efficient substrate. In support of these absence of Dbf4 protein, Cdc7 protein was still able to co- findings, similar phosphorylation of GST-Rev7 was observed immunoprecipitate with the Rev7-Myc13 protein (Figure using purified recombinant Xenopus DDK (Takahashi and 6A). Co-immunoprecipitation of Rev7-myc13 with Cdc7 Walter 2005) (Figure 5B). appears to be less efficient when Dbf4 was absent as more Rev7-myc13 remains in the supernatant when Dbf4 is ab- Rev7 protein binds to Cdc7 protein in the absence sent (Figure 6A). However, using plasmid pYQ118 (Table 2; of Dbf4 HA-Dbf4) in cdc7D mcm5-bob1 strain (yLPB163; Table 1) Because an inactive kinase-dead Cdc7 kinase can still bind that contains a tagged REV7-MYC13 gene, we found the re- Rev7 protein (Figure 3), we tested whether Cdc7 kinase is verse is not true; Dbf4 protein cannot co-immunoprecipitate able to bind to Rev7 in the absence of Dbf4 protein (Figure with Rev7-Myc13 in the absence of Cdc7 protein (Figure 6A). Because DBF4 and CDC7 are essential genes, we used 6B). We estimate the amount of Rev7-Myc13 protein co-

1116 L. N. Brandão et al. 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 Figure 5 Phosphorylation of yeast GST-Rev7 by yeast and Xenopus DDK. with HA-Cdc7 protein is only reduced 1.3-fold when Dbf4 A total of 2–4 mg of the indicated GST-tagged yeast protein were in- protein is absent. We conclude Rev7 protein binding of DDK cubated with (A) Cdc7-Myc immunopreciptates from strain YJO235 or (B) Xenopus 32 is mediated primarily by the Cdc7 kinase subunit and does 10 mgof DDK (xCdc7-xDbf4) in the presence of g P-ATP. Reactions were subjected to SDS–PAGE, proteins were transferred to not require the Dbf4 subunit, although Dbf4 protein likely nitrocellulose, and protein phosphorylation was visualized by autoradiog- contributes to stabilize the binding. raphy. Lanes in A were from different gels and aligned by the molecular weight standards. Anti-GST (UBI) blots of the filters showed the position Loss of DDK is not epistatic with the loss of PCNA post- fi fi and con rmed equivalent amounts of the indicated GST fusion proteins translational modi cations and Pol32 in survival (not shown). (A) Lane 1, Cdc7-Myc IP plus GST-Mcm2 (103 kDA); lane 2, following UV irradiation 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 We investigated whether DDK involvement in TLS was re- Cdc7-Myc IP in lanes 2–4. (B) Lane 1, Xenopus DDK only; lane 2, GST gulated by interacting with subunits of Pold that are impor- only; lane 3, GST-Mcm2 only; lane 4, GST-Rev7 only; lane 5, GST plus tant in Polz-dependent mutagenesis using genetic epistasis DDK; lane 6, GST-Mcm2 plus DDK; lane 7, GST-Rev7 plus DDK. Note DDK analysis (Figure 7) (Boiteux and Jinks-Robertson 2013). Polz autophosphorylation of both xDbf4 and xCdc7 subunits. also interacts with Pol32, a nonessential subunit of Pold that is required for Pol z-dependent mutagenesis (Gerik et al. 1998; least an additive and possible synergistic interaction be- Huang et al. 2000). In biochemical analyses, Polz holoenzyme tween pol30-K127R,K164R and cdc7D mcm5-bob1 at three is a four subunit complex called Polz4 that contains Pol32 different UV doses (Figure 7A, Table 3). Furthermore, and Pol31 in addition to the core Rev3 and Rev7 proteins pol32D also showed a nonepistatic relationship with cdc7D (Makarova et al. 2012). Polz also interacts with PCNA mcm5-bob1 (Figure 7B, Table 3). We also examined possible (encoded by POL30), a homotrimeric sliding clamp that pro- physical interactions of Cdc7 and Dbf4 with GST-Rev1, motes DNA polymerase processivity (Ayyagari et al. 1995). Pol32-HA, or FLAG-Pol30 proteins using co-IP and GST PCNA is modified at K127 and K164 by sumoylation and pulldown experiments but failed to detect any interactions ubiquitination, respectively, to modulate postreplication repair (Pessoa-Brandão 2005). (PRR) pathways (reviewed in Boiteux and Jinks-Robertson 2013). Monoubiquitination of PCNA by Rad6-Rad18 promotes Discussion TLS by Polz and Polh;thepol30-K164R and pol30-K127R, K164R alleles are epistatic with rev3D for survival following In this report, we have investigated the role of DDK in Polz- UV irradiation (Stelter and Ulrich 2003). Polyubiquitination of dependent TLS and mutagenesis. Previous studies showed monoubiqutinated PCNA by Ubc13-Mms2-Rad5 promotes DDK regulates mutagenesis induced by various DNA error-free damage avoidance (Hoege et al. 2002). damaging agents (Figure 1) (Njagi and Kilbey 1982; Given the epistasis between cdc7D and rev3D with respect Hollingsworth et al. 1992; Ostroff and Sclafani 1995; Pes- to survival following UV treatment, we examined whether soa-Brandão and Sclafani 2004), but DDK had not been the pol30-K127R,K164R or pol32D allele is similarly epistatic similarly implicated in spontaneous mutagenesis (Sclafani to a cdc7 null mutation. Instead of epistasis, we observed at 2000). Because DDK and Polz are in the same DNA-

DDK Regulates Polz and Mutagenesis 1117 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 Jinks- Robertson 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 Figure 6 Analysis of the co-IP of Rev7 by each subunit of DDK. Cell et al. 2010). However, even though MCM proteins are known extracts were incubated with anti-HA antibody, and precipitated proteins DDK substrates during replication (Sclafani and Holzen were separated by SDS–PAGE. Western blots were probed individually 2007), DDK phosphorylation of Mcm2 protein in vitro is not with an anti-HA or anti-Myc antibody to detect tagged proteins in the reduced (Oshiro et al. 1999) and phosphorylation of the immunoprecipitate. (A) WT strain YSS13 (lanes 1–5) or dbf4D strain RSY1445 (lanes 6–10) with HA-Cdc7 plasmid pCH766 was used. (B) Mcm7 protein is only modestly reduced during checkpoint WT strain YSS13 (lanes 1–5) or cdc7D strain yLPB163 (lanes 6–10) with activation (Weinreich and Stillman 1999). Furthermore, HA-Dbf4 plasmid pYQ118 was used. Lanes are labeled the same in A and yeast Mcm4 is still phosphorylated during DNA replica- B. Lane 1, input extract only; lane 2, supernatant of mock IP with no tion-checkpoint arrest in vivo (Sheu and Stillman 2006). antibody; lane 3, supernatant of IP; lane 4, pellet of mock IP; lane 5, pellet Recently, it was shown that DDK phosphorylation of yeast 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, Eco1, which is required to establish chromosome cohesion, pellet of IP; 20% of the IP was used for SDS–PAGE as compared to 5% of is inhibited by Rad53 phosphorylation of Dbf4 (Lyons et al. the supernatants or the input extract. 2013). Similarly, DDK phosphorylation of Mer2 during meiosis is inhibited by Rad53 phosphorylation of Dbf4 damage response pathway (Pessoa-Brandão and Sclafani (Blitzblau and Hochwagen 2013). Yet, in human cells, 2004) and Polz is required for spontaneous mutagenesis, DDK phosphorylates both Claspin (Kim et al. 2008) and the role of DDK in spontaneous mutagenesis was investi- hMcm2 (Tenca et al. 2007) during DNA damage both gated using the sensitive lys2DA746 reversion assay (Harfe in vivo and in vitro. Therefore, DDK is active during check- and Jinks-Robertson 2000). Significantly, we found DDK is point responses depending on the system, substrate, and required for the production of complex frameshift events assay used. Based on the current report, we infer that (CINS), which are a distinctive mutational signature of Polz DDK must be active during DNA damage checkpoint activa- (Harfe and Jinks-Robertson 2000; Sabbioneda et al. 2005) tion to allow for TLS. Similarly, human DDK on chromatin (Figure 2). We conclude DDK is required for Polz-dependent remains active during checkpoint activation to facilitate spontaneous, as well as induced, mutagenesis. TLS by Polh (Yamada et al. 2013). It was proposed there In addition to a reduction in CINS, the cdc7D mcm5- are two distinct pools of DDK with the soluble pool bob1 strain also showed a reduction in the number of large inhibited to block late origin firing, while the chromatin- deletions that revert the lys2DA746 allele (Figure 2). bound pool remains active to facilitate TLS (Yamada et al. These large deletions have been attributed to a decrease 2013). It is also possible the effect of checkpoint activation in Pold processivity, as they increase in pol30-46 and on DDK function is either substrate specific(Blitzblauand pol32D mutants (Minesinger and Jinks-Robertson 2005). Hochwagen 2013; Lyons et al. 2013) or affects different The large deletions are thought to be produced when downstream effectors.

1118 L. N. Brandão et al. 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 Furthermore, we show Rev7 binds largely to the Cdc7 activation, human Rad18 is phosphorylated by DDK to kinase subunit and not to the Dbf4 regulatory subunit (Fig- produce a binding site for Polh during TLS at sites of ure 6). Although Dbf4 protein is required for Cdc7 kinase to replication fork stalling (Day et al. 2010; Yamada et al. phosphorylate substrates (Kitamura et al. 2011), and may 2013). However, the “S box” region of human Rad18 that bind substrates in the Zn++ binding domain of motif C is phosphorylated by human DDK is not conserved in (Hughes et al. 2012), it has not been determined whether either budding or ﬁssion yeast and it was suggested Cdc7 or Dbf4 protein alone can bind substrate proteins. In DDK in yeast may phosphorylate other TLS proteins contrast, the N-terminal domain of Dbf4 is required for DDK (Day et al. 2010). In support of this idea, we show in this to bind to MCM2–7 complex in the pre-replication complex report that Rev7 subunit of Polz maybethetargetasDDK in vivo (Duncker et al. 2002; Varrin et al. 2005) and in vitro binds to (Figure 3) and phosphorylates Rev7 protein (Sheu and Stillman 2006; Francis et al. 2009). In cyclin- (Figure 5). Therefore, there is conservation of DDK func- dependent kinases (CDKs), the main recognition site for the tion as DDK regulates TLS polymerases in both humans substrate is in the CDK subunit, while the cyclin hydropho- and yeast. bic patch stabilizes the interaction and increases potential

DDK Regulates Polz and Mutagenesis 1119 Table 3 Mathematical analysis of UV survival data

a Expected 2ln Sdm 2 a Strain–genotype UV dose (J/m ) (for additive interaction) Observed 2ln Sdm yLPB226 cdc7D pol30-K127, 20 2.29 3.16 6 0.24 K164R 50 4.42 5.52 6 0.05 70 6.43 6.91 6 0.11 yLPB95 cdc7D pol32D 20 1.46 1.63 6 0.10 50 2.27 3.27 6 0.12 70 3.91 4.96 6 0.13 a 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 strain constructions. Marianne Tecklenburg provided excel- analogy, we propose Cdc7 protein can bind Rev7 without Dbf4 lent technical assistance. We thank Helle Ulrich (Max Planck unlike binding to the Mcm4 substrate, which requires Dbf4. Institute), Chris Hardy (Vanderbilt University) and Marco Although there are a number of possible interpretations Muzi-Falconi (University of Milan) for strains and plasmids. of additive and synergistic interactions with DNA repair This work was supported by Public Health Service awards to pathways (Brendel and Haynes 1973), one interpretation of R.A.S. (R01 GM35078) and to S.J.-R. (R01 GM64769), the interaction seen between cdc7D and pol32D or pol30- a National Research Service Award fellowship to R.F. K127R,K164R mutations (Figure 7, Table 3) is Pol32 protein (F32GM100644) and an Institutional Research and Career and the Pol30 (PCNA) modifications regulate multiple TLS Development Award to I.S. (K12 GM000680). L.N.B. was and also replicative polymerases (Boiteux and Jinks-Robertson supported by a National Institutes of Health training grant 2013), while DDK regulates TLS differently by regulating Polz (T32 GM008730). through regulation of Rev7.DDKmayregulateTLSlesion bypass during “gap-filling” rather than during “polymerase switching” (Waters et al. 2009). In the gap-filling model, rep- Literature Cited riming of replication occurs downstream of the lesion leaving a gap, which is subsequently filledinbyTLS.Thisisconsistent Ayyagari, R., K. J. Impellizzeri, B. L. Yoder, S. L. Gary, and P. M. Burgers, 1995 A mutational analysis of the yeast proliferating with the pol30 mutant being more sensitive to UV than the cell nuclear antigen indicates distinct roles in DNA replication cdc7 mutant (Figure 7). and DNA repair. Mol. Cell. Biol. 15: 4420–4429. Previously, we have shown Cdc7 kinase activity to be re- Blitzblau, H. G., and A. 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