g model article in pressmicrobiology.ucdavis.edu/kowalczykowski/pdf_files/siebler et al, 201… ·...

D

ArS

HE

a

ARRAA

KPPMTPU

1

ebtcv

t

((

h1

ARTICLE IN PRESSG ModelNAREP-1952; No. of Pages 12

DNA Repair xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

DNA Repair

j ourna l ho me pa ge: www.elsev ier .com/ locate /dnarepai r

novel variant of DNA polymerase �, Rev3�C, highlights differentialegulation of Pol32 as a subunit of polymerase � versus � inaccharomyces cerevisiae

ollie M. Siebler, Artem G. Lada, Andrey G. Baranovskiy, Tahir H. Tahirov, Youri I. Pavlov ∗

ppley Institute for Research in Cancer and Allied Diseases, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA

r t i c l e i n f o

rticle history:eceived 7 February 2014eceived in revised form 17 April 2014ccepted 24 April 2014vailable online xxx

eywords:olymerase �ol32gs1

ranslesion DNA synthesisolymerase switchV mutagenesis

a b s t r a c t

Unrepaired DNA lesions often stall replicative DNA polymerases and are bypassed by translesion syn-thesis (TLS) to prevent replication fork collapse. Mechanisms of TLS are lesion- and species-specific,with a prominent role of specialized DNA polymerases with relaxed active sites. After nucleotide(s) areincorporated across from the altered base(s), the aberrant primer termini are typically extended by DNApolymerase � (pol �). As a result, pol � is responsible for most DNA damage-induced mutations. Themechanisms of sequential DNA polymerase switches in vivo remain unclear. The major replicative DNApolymerase � (pol �) shares two accessory subunits, called Pol31/Pol32 in yeast, with pol �. Inclusionof Pol31/Pol32 in the pol �/pol � holoenzymes requires a [4Fe–4S] cluster in C-termini of the catalyticsubunits. Disruption of this cluster in Pol � or deletion of POL32 attenuates induced mutagenesis. Herewe describe a novel mutation affecting the catalytic subunit of pol �, rev3�C, which provides insight intothe regulation of pol switches. Strains with Rev3�C, lacking the entire C-terminal domain and thereforethe platform for Pol31/Pol32 binding, are partially proficient in Pol32-dependent UV-induced mutagen-esis. This suggests an additional role of Pol32 in TLS, beyond being a pol � subunit, related to pol �. Insearch for members of this regulatory pathway, we examined the effects of Maintenance of Genome
Stability 1 (Mgs1) protein on mutagenesis in the absence of Rev3–Pol31/Pol32 interaction. Mgs1 maycompete with Pol32 for binding to PCNA. Mgs1 overproduction suppresses induced mutagenesis, buthad no effect on UV-mutagenesis in the rev3�C strain, suggesting that Mgs1 exerts its inhibitory effectby acting specifically on Pol32 bound to pol �. The evidence for differential regulation of Pol32 in pol �and pol � emphasizes the complexity of polymerase switches.
© 2014 Elsevier B.V. All rights reserved.

. Introduction

Cellular DNA is under constant attack by exogenous andndogenous mutagens. Resulting lesions, if unrepaired [1,2], canlock cell division when replicative DNA polymerases are unable

Please cite this article in press as: H.M. Siebler, et al., A noferential regulation of Pol32 as a subunit of polymerase �

http://dx.doi.org/10.1016/j.dnarep.2014.04.013

o incorporate nucleotides across from the damaged sites. Thisauses activation of DNA damage tolerance mechanisms to pre-ent irreversible replication fork collapse and to finish replication

Abbreviations: CTD, C-terminal domain; FeS, [4Fe–4S] iron sulfur; Mgs1, Main-enance of Genome Stability 1; ZnF, zinc finger.∗ Corresponding author. Tel.: +1 402 559 7717; fax: +1 402 559 8270.

E-mail addresses: [email protected] (H.M. Siebler), [email protected]. Lada), [email protected] (A.G. Baranovskiy), [email protected]. Tahirov), [email protected], [email protected] (Y.I. Pavlov).

ttp://dx.doi.org/10.1016/j.dnarep.2014.04.013568-7864/© 2014 Elsevier B.V. All rights reserved.

of the genome [3]. The DNA damage tolerance pathway includespredominantly error-free recombinational damage avoidance andtranslesion synthesis (TLS), which often is a source of mutations[4,5]. While TLS machinery deals with the lesion, the replicationfork can restart downstream to allow for continuation of replication[6].

During TLS, replicative DNA polymerases yield the dam-aged template to specialized polymerases which incorporatenucleotides across from the altered base(s) [7–9]. Most prominentin TLS are the low fidelity Y-family polymerases �, �, �, andRev1 [10,11], but in some cases insertion is accomplished byX-family, A-family, or B-family DNA polymerases [5,12]. Then

vel variant of DNA polymerase �, Rev3�C, highlights dif-versus � in Saccharomyces cerevisiae, DNA Repair (2014),

there is extension of the aberrant primer terminus, achieved bythe inserter or another polymerase. Most frequently this extensionis accomplished by the error-prone B-family polymerase � (pol�) [4,12–14]. Once the lesion is bypassed, there is a return to

dx.doi.org/10.1016/j.dnarep.2014.04.013


http://www.sciencedirect.com/science/journal/15687864

http://www.elsevier.com/locate/dnarepair

mailto:[email protected]







IN PRESSG ModelD

2 A Repair xxx (2014) xxx–xxx

sto

Tatibmtieaa[

basemtpacirpswfu

ristororolTtic

[cp(shtfsb[Tntsld

Table 1Nomenclature for yeast and human Pol � and Pol �.

Organism Subunit Gene Protein

Polymerase ıYeast Catalytic POL3 Pol3

B POL31 Pol31C POL32 Pol32

Human Catalytic POLD1 p125B POLD2 p50C POLD3 p66Small 4th POLD4 p12

Polymerase �Yeast Catalytic REV3 Rev3

Accessory REV7 Rev7B, C POL31, POL32 Pol31, Pol32

ARTICLENAREP-1952; No. of Pages 12

H.M. Siebler et al. / DN

ynthesis by replicative polymerases or filling of the gap betweenhe bypassed lesion and a downstream restart site, by pol � itselfr by replicative polymerases [5,6].

TLS events can have opposing effects on mutagenesis. SomeLS polymerases are tailored to bypass specific types of lesionsnd incorporate predominantly the correct base, i.e. the basehat should have been incorporated by the replicative polymerasen the absence of damage. Historically, this is called error-freeypass because the action of these polymerases suppresses inducedutagenesis. However, the number of lesions greatly exceeds

he number of polymerases. Therefore most lesions are primar-ly bypassed by the addition of an incorrect base. This so-calledrror-prone TLS is highly mutagenic. This process is carried out by

complex of proteins composed of replicative pols, TLS pol �, Rev1,nd monoubiquitylated proliferating cell nuclear antigen (PCNA)4,9,15,16].

One critical event during TLS in eukaryotes is the physical switchetween the polymerases. Details of how it actually occurs in vivore not clear. Currently it is thought that it occurs via the two-tep insertion–extension mechanism, proposed on the basis ofxperiments in yeast (Fig. 1A) [11,12,17]. Upon damage, PCNA isonoubiquitylated at K164 [18] and there is a switch from replica-

ive pol � (or pol �) to another polymerase (predominantly Y-familyol) which inserts a nucleotide across from the lesion. Rev1 acts asn indispensable scaffold protein and, when necessary, a deoxy-ytidyl transferase inserting “C” opposite the lesion. Then theres a switch to pol � which performs extension from this aber-ant terminus. If an error was made during bypass, the action ofol � allows the altered sequence to remain in the nascent DNAtrand sequence, leading to a mutation. Malfunction of this path-ay abolishes induced mutagenesis. The signals involved, aside

rom ubiquitylation and probably deubiquitylation of PCNA, arenknown [19,20].

Pol � is responsible for most induced point mutations andoughly half of spontaneous mutations [9,21]. It synthesizes DNAn vitro with low fidelity and produces a characteristic mutationalignature [22], found in mutation spectra in vivo [2,23,24]. Part ofhe signature is attributed to template switches [25]. Pol � is thenly TLS polymerase essential for viability in mice, suggesting it isequired for tolerance of endogenous DNA damage during devel-pment. In yeast, deletion of REV3 is not lethal but causes growthetardation in strains with elevated levels of abasic sites [26]. Lossf the catalytic subunit of pol � or Rev1 results in elevated rates ofarge deletions [24,25] and gross chromosomal abnormalities [27].herefore, while error-prone TLS is etiologic in most environmen-ally induced cancers, its absence can also contribute to genomenstability and cancer [13,28,29]. Pol � can also contribute to cancerell resistance to the chemotherapeutic agent cisplatin [30].

Pol � was long thought to be composed of only Rev3 and Rev731]. We discovered that the C-terminal domain (CTD) of the humanatalytic subunit of pol � binds two accessory subunits of pol �,50/p66, and predicted that human pol � is a four-subunit complexSee Table 1 for nomenclature of human and yeast DNA polymeraseubunits) [32]. Four-subunit human pol � was later purified fromuman cells and possessed polymerase activity superior to thewo-subunit enzyme [33]. Yeast pol � can also stably exist as aour-subunit enzyme, containing the catalytic subunit Rev3, acces-ory subunit Rev7, and Pol31/Pol32 [34–36]. In this complex, Pol32inds to Pol31, and Pol31 binds to the CTD of catalytic subunit Pol337–39]. The existence of shared subunits between replicative andLS pols was the basis for the proposal of an additional mecha-ism of switching between pol � and pol � through an exchange of



he catalytic subunits on Pol31/Pol32 bound to PCNA [32]. In thiscenario (Fig. 1B), pol � stalling at a lesion signals for monoubiquity-ation of PCNA. Then the catalytic subunit Pol3 dissociates (and/or isegraded [40]) and Rev3/Rev7 is recruited to Pol31/Pol32 left at the

Human Catalytic REV3L p353Accessory REV7 P30B, C POLD2, POLD3 P50, p66

site of the lesion. This mechanism provides an easy, yet unproven,possibility for a switch back to Pol3 for processive synthesis if nec-essary (more in Section 4, Discussion). In this model, pol � plays arole in TLS by regulating the entire switch process.

It is believed that based on the structure of another B-familymember pol � and a low resolution EM structure of pol �, that bothPol3 and Rev3 contain a CTD attached by a flexible linker [39,41].Both polymerases contain a FeS cluster in this domain [42], whichis required for binding to Pol31/Pol32 [32,34,35]. In addition, whenthe C-terminal tail of Rev3 past the metal binding sites is removedthere is no binding to Pol31, suggesting that not only is the clus-ter necessary for binding but the region of the CTD downstream ofit is also [34]. In yeast when the FeS cluster of pol � is disrupted,there is a severe reduction of mutagenesis comparable to the com-plete absence of Rev3 ([32,34], present study). It is possible thatthis cluster plays a structural role or that the switch is regulatedby oxidation–reduction reactions [43]. To better understand howpolymerase switches occur in yeast, we created several mutantsaffecting the CTD of Rev3 (Fig. 2A). Intriguingly, even though disrup-tion of the cluster nearly eliminated induced mutagenesis, a mutantthat lacked the entire CTD and thus the whole platform for interac-tion with Pol31/Pol32, exhibited robust mutagenesis at low doses ofUV irradiation and residual mutagenesis at higher doses. The pur-pose of this study was to characterize this C-terminal truncationmutant, rev3�C, and use it as a tool to probe our understanding ofpolymerase switches in vivo.

We found that UV-induced mutagenesis in this mutant stillrequires the presence of Pol32 but becomes independent of reg-ulation by Maintenance of Genome Stability 1 (Mgs1), whoseoverproduction suppresses mutagenesis in strains with normal pol� [44]. We conclude that Pol32 plays a role in mutagenesis beyondits function as subunit of pol � and that Mgs1 is capable of regulat-ing UV-induced mutagenesis only when the Pol31/Pol32 bindingplatform in Rev3 is intact.

2. Materials and methods

2.1. Materials

Most mutagenesis studies were done in the Saccharomycescerevisiae strain 8C-YUNI101 (MATa his7-2 leu2-3,112 ura3-� bik1::ura3-29RL trp1-1UAG ade2-1UAA) [45] and its deriva-tives. Mutagenesis studies on the deletion of MGS1 weredone in a derivative of the strain BY4742 (MAT ̨ his3�1


leu2�0 lys2�0; ura3�0) (Life Technologies, USA). Extracts forwestern blotting were prepared from transformants of theprotease-deficient strain BJ2168 (MATa prc1-407 prb1-1122 pep4-3 leu2 trp1 ura3-52 gal2) [46]. Plasmids used are described


Please cite this article in press as: H.M. Siebler, et al., A novel variant of DNA polymerase �, Rev3�C, highlights dif-ferential regulation of Pol32 as a subunit of polymerase � versus � in Saccharomyces cerevisiae, DNA Repair (2014),http://dx.doi.org/10.1016/j.dnarep.2014.04.013

ARTICLE IN PRESSG ModelDNAREP-1952; No. of Pages 12

H.M. Siebler et al. / DNA Repair xxx (2014) xxx–xxx 3

Fig. 1. Models of polymerase switching during TLS. (A) Classic two-event polymerase switch model. There is a switch from pol � to a TLS complex composed of a Y-familypol such as Rev1 or pol � and pol �. After insertion of a nucleotide opposite the lesion by a member of this complex, pol � extends the primer. Rev1 also acts as a scaffold.(B) Illustration of the switch from pol � to pol � in the new variant of polymerase switch model utilizing the exchange of Pol31/Pol32 subunits. The catalytic subunit of pol �(Pol3) dissociates from the DNA and Rev3/Rev7 binds to the Pol31/Pol32 still left on DNA. Other steps are the same as in A.

Fig. 2. Effects of rev3 mutations on UV mutagenesis alone and in the presence of wild-type REV3. (A) Schematic view of Rev3 variants used in this study. (B) WT strainsadditionally expressing mutant variants of Rev3 exhibit WT levels of survival and mutagenesis. Strains possess WT REV3 in the chromosomal location and mutant rev3 on anexpression plasmid. WT + vector (pink �), rev3�C + vector (orange �), rev3-FeS + vector (green �), WT + rev3-dd↑ (blue �), WT + rev3�C↑ (black ♦), WT + rev3-FeS↑ (purple �).



4 H.M. Siebler et al. / DNA Repair xxx (2014) xxx–xxx

Table 2Description of mutant strains used in this study.

Relevant genotypea Principal defect or combination Comments

REV3 mutants rev3-dd Catalytically inactive (D1142A, D1144A) pCAV2 plasmid was subjected to sitedirected mutagenesis in the Dr. M. Diazlaboratory (NIEHS) and when mutationwas transferred in genomic location of8C-YUNI101, yeast strain was UVimmutable (personal communication)

rev3-FeS Disruption of FeS cluster binding site (C1446A, C1449A,C1468A, C1473A)

This site was called MBS2 and mutantfirst described in [32]

rev3�C Absence of Rev3 C-terminal domain (amino acids1381–1505)

This study

Other mutants pol30-K164R PCNA (encoded by POL30 gene) variant that cannot beubiquitylated (K164R) [18]

Plasmid to create this mutantdescribed in [23]

pol32� Absence of Pol32 due to deletion of POL32 gene, leads toimmutability [51]

Disruption by kanMX cassette

rev1� Absence of Rev1 due to deletion of REV1 gene Disruption by kanMX cassettemgs1� Absence of Mgs1 due to deletion of MGS1 gene Disruption by kanMX cassetteMGS1↑ Overexpression of MGS1 under the GAL1 promoter

(GAL1–MGS1 integrated at LEU2)Constructed in this work by integrationof the plasmid YIp128-GAL-MGS1, asdescribed in [44]

Double mutants rev3�C-dd Combination of rev3�C and rev3-dd This studyrev3�C pol32� Combination of rev3�C and pol32� This studyrev3�C rev1� Combination of rev3�C and rev1� This studyrev3�C mgs1� Combination of rev3�C and mgs1� This studyrev3�C MGS1↑ Overexpression of MGS1 in the rev3�C strain This studyrev3�C pol30-K164R Combination of rev3�C and pol30-K164R This study

Strains with plasmidsb WT + vector 8C WT strain with pRS425-GALGST empty vector This studyWT + REV1↑ REV1 overexpression in WT strain with

pRS425-GALGST-REV1This study

WT + rev3-dd↑ rev3-dd overexpression in WT strain with Yep181-rev3-dd This studyWT + rev3�C↑ rev3�C overexpression in WT strain with

pRS425-GALGST-rev3�CThis study

WT + rev3-FeS↑ rev3-FeS overexpression in WT strain withpRS425-GALGST-rev3-FeS

This study

rev3� + vector rev3� strain with pRS425-GALGST empty vector This studyrev3� + rev3�C↑ rev3�C overexpression in rev3� strain with

pRS425-GALGST-rev3�CThis study

rev3�C + vector rev3�C strain with pRS425-GALGST empty vector This studyrev3�C + REV1↑ REV1 overexpression in rev3�C strain with


rev3-FeS + vector rev3-FeS strain with pRS425-GALGST empty vector This studyrev3-FeS + REV1↑ REV1 overexpression in rev3-FeS strain from


rev1� + vector rev1� strain with pRS425-GALGST empty vector This studyrev1� + REV1↑ REV1 overexpression in rev1� strain from

V1This study

n 2).

iawaBl(cmDAS

2

Tgrfd

pRS425-GALGST-RE

a All mutants except mgs1� were created in the 8C-YUNI101 background (Sectiob Description of plasmids is in Section 2.2.

n the next section. Mouse anti-GST, goat HRP-conjugatednti-mouse, and donkey HRP-conjugated anti-goat antibodiesere from Genscript (Piscataway, NJ). Goat anti-human actin

ntibody (cross-reacts with yeast actin) was from Santa Cruziotechnology (Santa Cruz, CA). Super Signal West Femto Chemi-

uminescent Substrate detection kit was from Thermo ScientificDubuque, IA). The 1× complete EDTA-free protease inhibitorocktail was from Roche (Indianapolis, IN). The Immobilon PVDFembrane was from Millipore (Billerica, MA). QuikChange Siteirected Mutagenesis kit was from Agilent Technologies (USA).ll other chemicals were reagent grade and were purchased fromigma–Aldrich (St. Louis, MO) or Fisher Scientific (Atlanta, GA).

.2. Creation of mutants for this study

All mutant strains used in this study are listed in Table 2.he plasmid pRevLCav2-rev3�C is a deletion derivative of inte-



rative plasmid pRevLCav2 [45] created by PCR of the plasmidegion flanking the deletion and ligation, where rev3�C encodesor Rev3 lacking the C-terminus (amino acids 1381–1504). Siteirected mutagenesis with the QuikChange kit was further

used on pRevLCav2-rev3�C to create pRevLCAV2-rev3�C-DD,encoding for a catalytically dead Rev3. We used a standardintegration–excision protocol to integrate the mutant alleles intothe genome and replace endogenous REV3 [45], creating the rev3�Cand rev3�C-dd mutant stains, respectively (Table 2). These plas-mids were linearized with SnaBI before transformation. 8C WTand rev3�C strains were transformed with the BstEII-linearizedplasmid YIp128-GAL-MGS1 (kindly provided by H. Ulrich) to cre-ate WT + MGS1↑ and rev3�C + MGS1↑ strains, respectively, withintegration of the GAL-MGS1 cassette into the LEU2 locus. rev3�Cpol32� and rev3�C rev1� were created by replacing POL32 andREV1, respectively, with KanMX cassettes in the rev3�C strain.Strains for overexpression of rev3�C and REV1 (Table 2) wereobtained by transformation of 8C-YUNI101 rev3� and 8C YUNI101,respectively, with derivatives of the multicopy plasmid pRS425-GALGST [47] containing the appropriate GST-REV3 or GST-REV1allele under the control of a galactose-inducible promoter (plas-


mids with wild-type genes were constructed by N. Sharma and P.Shcherbakova, personal communication). The derivatives of p425-GALGST-REV3 plasmid with mutations affecting metal binding siteof Rev3 were created by gap repair in yeast in vivo and are first


ING ModelD

A Repa

dcg

2f

oaBoplo6Snwgqpafwo[wr

pgwdwp

wgea

2

tespmawgpNPvb5(ta

SaH



escribed in [32]. The plasmid for overexpression of rev3�C wasreated by the same method. All plasmids and site-directed muta-enesis primer sequences are available upon request.

.3. Determination of survival and induced mutagenesisrequencies

Yeast strains were typically grown for two days at 30 ◦C in 5 mLf yeast extract peptone dextrose medium with 60 mg/L adeninend uracil (YPDAU) with shaking. Cells were pelleted at 1000 × g in aeckman Model TJ-6 centrifuge for 2 min and re-suspended in 1 mLf sterile water. Cells were diluted 200,000-fold and 100 L aliquotslated on Synthetic Complete (SC) medium; 50–100 L of undi-

uted cells were plated on SC medium supplemented with 60 mg/Lf l-canavanine (Can). Plates were irradiated with 0, 20, 40, or0 J/m2 of UV light. After three days of growth at 30 ◦C, colonies onC plates were counted and survival was calculated by dividing theumber of colonies at each UV treatment by the number of coloniesithout exposure (independently for each strain). After five days of

rowth, colonies on Can plates were counted and the mutant fre-uency was calculated by dividing the number of colonies on Canlates at each UV dose by the number of colonies on the SC platet the same dose (SC colony count was first multiplied by dilutionactor) as described in [48]. The induced Canr mutant frequencyas calculated by subtracting the spontaneous frequency (with-

ut treatment) from the mutant frequency for each UV-light dose48]. All data points are averages of at least two independent trialsith duplicates of each sample in each trial. Error bars in Figs. 2–5

epresent standard deviation.Strains for overexpression studies of MGS1 contained a GAL1

romoter upstream of MGS1 integrated at the LEU2 locus. Muta-enesis studies in these strains were performed as described aboveith the following modifications. These strains were grown for twoays in 4.5 mL SC-raffinose medium (no glucose, 3% raffinose) andere induced with 0.5 mL sterile 20% galactose for 2.5 h prior tolating.

Experiments with overexpression of rev3�C and REV1 (Table 2)ere done as described above except the transformants were

rown in SC-raffinose lacking leucine (-leu) to select for the pres-nce of a plasmid, induced for 2.5 h with galactose, and plated withppropriate dilutions on SC-leu and SC-leu-Can plates.

.4. Preparation of yeast extracts and western blot

For the analysis of levels of soluble Rev3 variants, we usedhe overexpression conditions because natural endogenous lev-ls of Rev3 are very low. Transformants of protease-deficienttrain BJ2168 with appropriate variants of pRS425-GALGST-REV3lasmids (Sections 2.1 and 2.2) were grown in 12.5 mL SCGL-leuedium overnight. The following day 62.5 mL of SCGL-leu was

dded. The third day, 62.5 mL of YPGLA medium was added and cellsere allowed to grow for 2.5 h. Finally, cells were induced with 2.5 g

alactose for 4 h, collected, and flash frozen. Yeast extracts wererepared in buffer containing 50 mM Tris–HCl (pH 7.5), 300 mMaCl, 1 mM EDTA, 10% sucrose, 10 mM -mercaptoethanol, 1 mMMSF, and 1× complete EDTA-free protease inhibitor cocktail. Theolume of buffer (in L) equals the millgrams of wet cells multipliedy 2.68. Yeast cells overproducing Rev3 were thawed, mixed with00 L of 0.5-mm glass beads, and lysed with a Disruptor GenieTM

six cycles, two min each) at 4 ◦C. The lysates were cleared by cen-rifugation using a MIKRO 200R centrifuge at 8000 × g for 15 minnd then 10,000 × g for 10 min, all at 0 ◦C.



For western blot analysis, proteins were separated on an 8%DS-PAGE Laemmli gel at 200 V, followed by a 1 h, 20 V transfer ton Immobilon PVDF membrane at 4 ◦C. Mouse anti-GST and goatRP-conjugated anti-mouse secondary antibodies were used to

PRESSir xxx (2014) xxx–xxx 5

detect the GST-Rev3 fusion protein. Goat anti-human actin anti-bodies, which cross-react with yeast actin, and donkey anti-goatHRP-conjugated secondary antibodies were used to detect actin asthe loading control. The blot was developed using the Super SignalWest Femto Chemiluminescent Substrate detection kit.

3. Results

3.1. Robust mutagenesis at low UV doses and substantial, residuallevels of mutagenesis at higher UV doses in the rev3�C strain

UV-induced mutagenesis is an effective readout for TLS in yeast.Pol � is required for all induced mutations and deletion of REV3or REV7 results in complete loss of UV-induced mutagenesis. Tobetter understand the role of Rev3, its subunits, and accessory pro-teins in TLS we examined parameters of UV-induced mutagenesisin several mutants affecting different parts of the protein (Fig. 2A,Table 3). The C-terminus of Rev3 contains two cysteine-rich metalbinding sites, CysA and CysB [49]. CysA forms a zinc finger (ZnF)and CysB coordinates a FeS cluster [32,42]. In the current study, anovel mutation of REV3 was created that encoded a protein lack-ing the entire CTD, thus completely removing both metal bindingsites and the platform for binding to Pol31/Pol32 [32,34]. It willbe further referred to as rev3�C (Fig. 2A). We used the popularcanavanine-resistance forward mutation assay, where mutationsof various types in the CAN1 gene in yeast confer resistance to thetoxic drug canavanine [50].

Mutagenesis was measured in various rev3 mutants after expo-sure to increasing doses of UV irradiation (Table 3). Disruption ofthe Zn finger motif of Rev3 had no effect on survival or mutagen-esis, as shown previously [32]. In contrast, disruption of the FeScluster alone or both metal-binding sites resulted in severe reduc-tion of mutagenesis and a drastic decrease in survival, similar tothe catalytically inactive enzyme, Table 3. These results are consis-tent with literature[32,34,35]. Intriguingly, we found that at lowUV doses rev3�C showed robust mutagenesis levels comparableto WT Rev3. At higher UV doses, it retained residual mutagene-sis even though it lacked the necessary elements (see above) tobind Pol31/Pol32, including the FeS cluster. Residual mutagene-sis was at intermediate dose about 60% and at highest dose about10% of WT levels. The observed mutant frequencies in treated anduntreated cultures are shown as an illustration of the magnitude ofUV-induced mutagenesis (Table 3).

3.2. rev3 mutations do not exert a dominant negative phenotypein UV-induced mutagenesis

The fact that strains with Rev3�C and Rev3-FeS, both lackingthe binding platform for Pol31/Pol32, show different phenotypeswas intriguing. To further characterize these mutants, we testedwhether rev3 mutations lead to a dominant negative effect instrains with WT Rev3. We found that the presence of rev3�C, rev3-FeS, or rev3-dd on the expression plasmids (Section 2) in the wildtype strain (8C-YUNI101, see Table 2 and Section 3.3 for descriptionof rev3-dd mutant) did not affect UV-induced mutagenesis (Fig. 2B).Therefore, all rev3 mutants examined were not dominant negativeand equally unable to compete with WT pol �.

3.3. Residual mutagenesis in rev3�C strains is dependent on thecatalytic activity of Rev3

Two aspartates in the invariant DTD motif in region I of active


site of all B-family DNA polymerases are involved in catalysis andtheir substitution to alanines results in catalytic dead enzymes [9].To rule out the possibility that another polymerase is recruited toassist Rev3�C and is responsible for the mutagenesis seen in the




Fig. 3. Induced mutagenesis in the rev3�C strain is dependent on the catalytic activity of Rev3 and is not increased by overproduction of Rev3�C. (A) The rev3�C-dd mutantwas hypersensitive and immutable, demonstrating that mutagenesis in rev3�C is dependent on the catalytic activity of Rev3. 8C WT (pink �), rev3-dd (green �), rev3�C(orange �), rev3�C-dd (blue �). (B) Western blot analysis of the overproduced CTD mutants of Rev3. There was only a slight increase in the levels of rev3�C over WT Rev3. (C)A T + vec( nden

rihc

rtificial, robust overexpression of rev3�C over WT has no effect on mutagenesis. Wblue �). Data were collected and analyzed as described in Section 2.3 (three indepe



ev3�C strain, a mutant was created that was both catalyticallynactive and lacked the CTD of REV3 (rev3�C-dd). This mutant wasypersensitive and UV-immutable, similar to the mutant with onlyatalytically inactive rev3-dd (Fig. 3A). This demonstrates that the

tor (pink �), rev3� + vector (green �), rev3�C + vector (orange �), rev3� + rev3�C↑t trials). All strains are described in Table 2.


residual mutagenesis seen in rev3�C strains is dependent upon thecatalytic activity of Rev3.

It is formally possible that the Rev3�C protein is more sta-ble than the wild-type (WT) holoenzyme and thus, being more


Please cite this article in press as: H.M. Siebler, et al., A novel variant of DNA polymerase �, Rev3�C, highlights dif-ferential regulation of Pol32 as a subunit of polymerase � versus � in Saccharomyces cerevisiae, DNA Repair (2014),http://dx.doi.org/10.1016/j.dnarep.2014.04.013



Fig. 4. UV induced mutagenesis in the rev3�C and WT strains is under similar genetic control. (A) Survival and mutagenesis of WT and rev3�C strains are both dependentupon monoubiquitylation of PCNA and the presence of Pol32. 8C WT (pink �), rev3�C (orange �), pol30-K164R (green �), rev3�C pol30-K164R (blue �), pol32� (black ♦),rev3�C pol32� (purple �). (B) Survival and mutagenesis are also dependent upon the presence of Rev1. 8C WT (pink �), rev3�C (orange �), rev1� (green �), rev3�C rev1�(blue �). (C) Overexpression of exogenous Rev1 does not elevate mutagenesis at high doses of UV light in WT and rev3�C strains. WT + vector (pink �), WT + REV1↑ (lightteal ©), rev3�C + vector (orange �), rev3�C + REV1↑ (green �), rev1� + vector (blue �), rev1� + REV1↑ (black ♦), rev3-FeS + vector (purple �), rev3-FeS + REV1↑ (red �). Datawere collected and analyzed as described in Section 2.3 (three independent trials). All strains are described in Table 2.




Fig. 5. rev3�C truncation mutant is insensitive to suppression of UV-induced mutagenesis by overexpression of MGS1. (A) Overexpression of MGS1 suppresses mutagenesisonly in the WT strain. 8C WT (pink �), rev3�C (orange �), MGS1↑ (green �), rev3�C MGS1↑ (blue �). (A) insert: PCR analysis confirms the correct integration of anMGS1expression cassette in WT and rev3�C strains. The forward primer had homology to the plasmid backbone sequence and the reverse primer had homology to theb – DN5 t on m( n Sect

amftFio

3P

ttAbUAda

eginning of the MGS1 gene; these primers amplify a region of about 1.3 kb. Lanes 1 – WT + MGS1↑ DNA, 6 – rev3�C + MGS1↑ DN(A). (B) Deletion of Mgs1 has no effecgreen �), rev3�C mgs1� (blue �). Data were collected and analyzed as described i

bundant, can participate in UV-induced mutagenesis despiteissing subunits. However, we did not detect any substantial dif-

erences in Rev3 protein levels in the soluble fraction of extracts ofhe different rev3 mutant and WT strains by Western blot (Fig. 3B).urthermore, artificial overexpression of rev3�C from a galactose-nducible promoter did not significantly increase survival or levelsf mutagenesis in rev3�C strain (Fig. 3C).

.4. Residual mutagenesis in the rev3�C strain is dependent onol32, monoubiquitylation of PCNA, and REV1

The third subunit of replicative DNA polymerase � (Pol32) andhe monoubiquitylation of the processivity clamp PCNA are knowno be required for UV-induced mutagenesis in yeast [18,51,52].ccordingly, in strains with the PCNA K164R variant that cannote ubiquitylated, we observed decreased survival and suppressed



V-induced mutagenesis in both WT and rev3�C strains (Fig. 4A).lso deletion of POL32 eliminated UV-induced mutagenesis andecreased survival in the WT strain. Interestingly, we found thatlthough rev3�C lacks the domain needed for binding Pol31 and

A ladder, 2 – blank PCR sample (no DNA added), 3 – 8C WT DNA, 4 – rev3�C DNA,utagenesis in WT and rev3�C strains. 8C WT (pink �), rev3�C (orange �), mgs1�

ion 2.3 (three independent trials). All strains are described in Table 2.

thus Pol32, Pol32 is still required for mutagenesis in rev3�C strain(Fig. 4A).

The Y-family DNA polymerase Rev1 binds to the Rev7 subunit ofpol � and to the processivity factor PCNA. These interactions couldfacilitate mismatch extension by pol � during TLS. [53]. Rev1 isthought to act as a scaffold for recruitment of Y-family pol � and pol� to stalled replication forks and therefore is indispensable for UV-induced mutagenesis [4]. Deletion of REV1 reduced survival andeliminated UV-induced mutagenesis in both our WT and rev3�Cstrains (Fig. 4B). It is possible that Rev1 is the only anchor holdingRev3�C to PCNA, since this variant of Rev3 has lost the platformfor interaction with Pol31/Pol32 and thus one major mode of inter-action with PCNA. In this case, an increase in the concentrationof Rev1 could elevate the chances of this backup interaction andincrease mutagenesis. To test this, the effects of overproduction ofRev1 were investigated. Overexpression of REV1 from a multicopy


plasmid under the control of a galactose-inducible promoter didnot affect survival and only slightly increased induced mutagene-sis at high UV doses. However, it increased mutagenesis to the samedegree in both WT and rev3�C strains (Fig. 4C).




Table 3rev3�C strain shows robust mutagenesis at low doses of UV irradiation and retains residual mutagenesis at high doses.

Strain UV treatment (J/m2) Percent survivala Mutant frequency (×10−6)b Induced mutantfrequency (×10−6)b

8C WT 0 100.0 ± 6.7 1 ± 0.4 –20 69.2 ± 0.9 87 ± 12 86 ± 1240 23.8 ± 3.0 299 ± 42 298 ± 4260 7.4 ± 7.0 701 ± 137 700 ± 137

rev3-dd 0 100.0 ± 8.8 0.8 ± 0.6 –20 4.6 ± 0.0 1.7 ± 1.7 1 ± 140 3.9 ± 0.0 1.9 ± 1.2 1 ± 160 2.0 ± 0.1 7.5 ± 5.4 7 ± 5

rev3�C 0 100.0 ± 8.6 0.8 ± 0.2 –20 20.6 ± 1.3 130 ± 9 129 ± 940 4.8 ± 1.7 168 ± 33 167 ± 3360 2.7 ± 0.6 65 ± 15 64 ± 15

rev3-FeS 0 100.0 ± 15.8 0.8 ± 0.2 –20 6.7 ± 0.4 38.8 ± 18.5 38 ± 1840 4.1 ± 0.1 7.4 ± 3.6 7 ± 360 2.5 ± 0.1 13.1 ± 11.0 12 ± 11

D

3

vwgMstw

4

fraBpfdtlcptdwmst

rdi[teFtmWt

a Values are mean ± SD in %.b Values are mean ± standard deviation (SD).ata are averages of four independent trials.

.5. Mgs1 is a negative regulator of pol � in UV mutagenesis

It is known that overexpression of the ATPase MGS1 lowers sur-ival and severely decreases MMS-induced mutagenesis in cellsith WT Rev3 [44]. We found the same effect for UV-induced muta-

enesis (Fig. 5A). Interestingly and unexpectedly, overproduction ofgs1 had no effect on survival or the residual levels of mutagenesis

een in rev3�C (Fig. 5A). MGS1 deletion had no effect on UV sensi-ivity or induced mutagenesis in WT or rev3�C strains, consistentith the literature ([54], Fig. 5B).

. Discussion and conclusions

DNA polymerase switching is essential for TLS. Recently it wasound that there is a much more intimate relationship betweeneplicative and TLS polymerases than had been imagined: humannd yeast pol � shares two genuine subunits with pol � [36,43].ased on this, we proposed that a switch can occur directly fromol � to � on the platform of two shared subunits (Fig. 1B, see Table 1or nomenclature) [32]. In this scenario, the catalytic subunit p125issociates when pol � is stalled by a lesion, leaving p50/p66 boundo PCNA. Rev3/Rev7 is recruited, binds to p50/p66, and bypasses theesion. It was hypothesized that the signaling here may involve ahange in the redox state of the FeS clusters of Pol3 and Rev3. It isossible that the resulting conformation change is recognized byhe proteolytic machinery responsible for the removal and degra-ation of the catalytic subunit of pol � [40]. The data in the currentork emphasize the role of pol � in initiation of pol switches inutagenic TLS and indicate that putative subunit exchange is con-

istent with only a fraction (ranging from 0% to 90%, depending onhe dose) of UV light induced mutations.

In this study we examined a novel mutation affecting pol �,ev3�C, which provides new insight into the regulation of pol �uring TLS. It has been shown that mutation of the FeS cluster

n the CTD of Rev3 severely decreases UV-induced mutagenesis32]. Intriguingly, the rev3�C mutant allele that encodes for a pro-ein with a deletion of the entire CTD (Fig. 2A) has a much milderffect (Table 3, Figs. 2B and 3B), despite the fact that this criticaleS cluster as well as a downstream region required for interac-



ion with Pol31/32 are absent [34,35]. We show here that neitherutant can act in a dominant negative fashion in the presence ofT Rev3 (Fig. 2B). This means that the different phenotypes of

he two mutants cannot be explained by different effects of the

two proteins on the TLS complex – in the presence of WT pol �they are both excluded from any transactions. The rev3�C yeaststrain shows robust mutagenesis at low UV doses and substantialmutagenesis at higher doses. This mutagenesis is dependent uponthe catalytic activity of pol � (Fig. 3A). This intermediate effect oninduced mutagenesis resembles effects of rev3 truncation mutantsthat retained the FeS cluster but lacked binding to Pol31/Pol32 [34].The effect indicates that the mechanisms of TLS might be somewhatdifferent at low and high doses of mutagens and depend on the levelof DNA damage.

We decided to further characterize the unexpected effect of ourrev3�C mutation and use it as a tool to probe the mechanisms ofTLS. It is known that deletion of any of the genes encoding for Rev3,Rev7, Y-family pol Rev1, or Pol32 results in loss of all induced muta-genesis [43]. We showed here that mutagenesis is abolished whenrev3�C is combined with an additional mutation causing a defectin the catalytic site of Rev3; therefore in vivo the Rev3�C/Rev7polymerase is catalytically active and is responsible for mutationgeneration. In further experiments we found that mutagenesis inrev3�C strains is dependent on monoubiquitylated PCNA, consis-tent with the data from literature for WT pol � (Fig. 4A).

For a long time it has not been well-understood why a subunit ofpol �, Pol32, is required for TLS. Now it is clear that Pol31/Pol32 arealso subunits of Pol �, though they appear to interact differently incomparison to the interaction with the catalytic subunit of pol �, asdiscussed in [35]. This observation led to a simple explanation forwhy Pol32-deficient cells are immutable: because Pol32 is a subunitof pol �. This is consistent with the fact that the FeS-less pol � variantlacks binding to Pol31/Pol32 and confers immutability. However,the data presented here suggest that the scenarios of polymeraseswitches are more elaborate and complex.

We have found that even though the protein in the rev3�Cmutant lacks the region required for binding to Pol31/Pol32, thestrain is still quite proficient in UV-induced mutagenesis. The effectof rev3�C is recessive, because it could be seen only when noWT REV3 was present in the genome (Fig. 2B). This is consistentwith observations that two-subunit pol � (Rev3 and Rev7) is activein vitro, albeit less active than four-subunit pol � [33,35]. Thus, Pol32is not critical for elementary pol � function. Despite the fact that


the rev3�C mutant most likely is not utilizing Pol32, we found thatmutagenesis in the rev3�C strain is absolutely dependent on Pol32.This strongly suggests that the immutability of pol32� strains atleast partly reflects an additional role of Pol32 in TLS related to pol


ING ModelD

1 A Repa

�mospwosu

DmpeePpimistbr

iibFemtd

iiRIwttud�cpctmTlfW

ietMgrh

in[t


0 H.M. Siebler et al. / DN

because the loss of this subunit of pol � prevents UV-inducedutagenesis independent of its binding to Rev3. This phenomenon

f cross-talk between replicative and TLS pols better fits the initialtep of switch model depicted in (Fig. 1B). It appears that Pol32-lessol � is unsuitable for a proper switch to pol �. This is consistentith the fact that the pol3-13 mutation, leading to a change of

ne cysteine involved in FeS binding in the CTD of pol �, leads touppression of UV mutability [55] and (Stepchenkova and Pavlov,npublished data).

It is generally assumed that pol � is involved only in laggingNA strand replication [56]. However, TLS events occur by sameechanism on both DNA strands in yeast system with damaged

lasmid [57]. In this context it is interesting that deletion of POL32liminates all mutagenesis, suggesting that Pol32 is required for TLSvents on both strands. Our data suggest that pol � is active withoutol32 and that the effect of pol32� is partly due to it being part ofol �. If pol � is fully responsible for the whole leading strand, then

t is difficult to explain why missing components of pol � attenuateutagenesis on this strand. This supports the idea that pol � is also

nvolved in leading strand replication [58] or that the polymerasewitch involves a complex event: pol � switching to pol � and theno pol �. Another possibility is that pol � is involved in mutagenesisy taking over synthesis from pol � or filling the gaps resulting frome-initiation of DNA synthesis after TLS downstream of pol � [5,6].

A recent paper suggested that Rev7 of pol � can bind to Pol32n vitro, which would indicate that Rev3�C could hold on to thisnteraction [39]. However, this interaction appears to be weakecause Rev7 was not pulled down with Pol32 by tagged Pol31 [34].urthermore if Rev7 were sufficient for binding to Pol32, we wouldxpect to see substantial levels of mutagenesis in the pol � FeSutant. This is not observed. Finally, mutations in POL31 abolishing

he interaction between Pol31 and Pol32 lead to UV-immutability,espite the fact that Pol32, per se, is untouched [37].

Another important member of the TLS machinery is Rev1. Itnteracts with Rev3 via Rev7 subunit [59]. Mutagenesis in rev3�Cs dependent on Rev1 which indicates that pol � with truncatedev3 is recruited by Rev1, a scaffold protein during regular TLS.

t is likely that the Rev1 interaction with pol � is the reason thate see intermediate mutagenesis in rev3�C. WT pol � can contact

he processivity clamp PCNA through both Rev1 and Pol31/Pol32,herefore there is robust TLS [60]. It is possible that rev3-FeS isnable to bind not only Pol31/Pol32 but also Rev1 due to steric hin-rance caused by the absence of the FeS cluster in the CTD of pol. Rev3�C lacks the CTD necessary for binding to Pol31/Pol32 butan still maintain its contact with Rev1, which is sufficient for sup-orting some TLS functions and for mutagenesis at low doses butonfers partial defect at higher doses. To explore this hypothesis, weested whether overproduction of Rev1 would increase UV-induced

utagenesis in rev3�C strains but found no such influence (Fig. 4C).herefore, simple increase of Rev1 levels cannot compensate for theack of subunits. It appears that chromosomal REV1 is sufficient toulfill the demand of the Rev1 protein after UV irradiation in both

T and rev3�C strains.In most of our experiments, the genetic control of mutagenesis

n the rev3�C strain was very similar to the WT strain. How-ver we found one modulator of TLS, encoded by the gene MGS1hat behaved differently. Overproduction of Mgs1 severely reduces

MS-induced mutagenesis [44] and eliminates UV-induced muta-enesis (Fig. 5A) in yeast, suggesting that it can act as a negativeegulator of TLS. However we found that overproduction of Mgs1ad no effect on mutagenesis in the rev3�C strain.

Mgs1 is an enigmatic regulator of TLS, an ATPase that plays a role



n maintaining genomic stability in yeast by an unknown mecha-ism. It is thought that Mgs1 helps maintain proper DNA topology61] and it is targeted to sites of replication stress through interac-ions with monoubiquitylated PCNA [44,62]. The human homolog

PRESSir xxx (2014) xxx–xxx

of Mgs1, Werner interacting protein 1 (WRNIP1), stimulates pol �in vitro and binds the catalytic subunit p125, p50, and p12 [63].Therefore it is plausible that Mgs1 binds pol � as well as pol �.

Deletion of MGS1 abrogates the growth defect of pol32�,pol3�ct, and pol31 mutant cells, suggesting that in the contextof damage sensitivity Mgs1 is an effector of Pol � [44,64]. It wasalso previously proposed that Mgs1 could compete with Pol32 forbinding to PCNA, as overproduction of Mgs1 reduced the yeast two-hybrid interaction between Pol32 and Ub-PCNA [44]. Therefore,it is possible that Pol32 could be knocked off of PCNA by Mgs1,resulting in lack of mutagenesis. However, we show here that over-expression of Mgs1 has no negative effect on the rev3�C strain. Thisresult suggests that in the context of UV-induced mutagenesis Mgs1exerts its inhibitory effect on mutagenesis by acting specifically onPol32 bound to pol �, not pol �. This is the first time that Mgs1 andpol � have been implicated to functionally interact. This result alsoargues against the idea that the interaction between pol � and Pol32is achieved through Rev7 binding to Pol32 (see above). If that werethe case, Rev3�C would still be bound to Pol32 and Mgs1 wouldcompete with it for binding, thus decreasing UV-induced muta-genesis in that strain. Consistent with the data in the literature, wefound that deletion of MGS1 had no effect on induced mutagenesis([54], Fig. 5B), which is also true for the rev3�C strain (Fig. 5B). Mgs1at normal physiological levels therefore may not play an active rolein induced mutagenesis.

This raises the question of why Mgs1 preferentially affectsinvolvement in mutagenesis of pol � and not pol �, since both con-tain Pol32. As mentioned, the nature of these interactions is notidentical. It was shown that Pol3 can form a stable complex withPol31 alone, but Rev3 cannot [35]. A recent EM structure of pol �may also give a clue to this differential binding nature [39]. Bothpol � and pol � contain catalytic and regulatory modules in theirstructures. However, there is flexibility between the two in pol �,whereas four subunit pol � appears to be more rigid. When Mgs1displaces Pol32 of pol �, the flexibility may result in only Pol32being temporarily displaced instead of the whole enzyme disso-ciating from PCNA. Because pol � is rigid, Mgs1 competition withPol32 results in the whole polymerase being removed from PCNA.

It has also been suggested that pols � and � interact with PCNAdifferently. This was shown most clearly with a mutation affect-ing in the monomer–monomer interface, pol30–113 [23]. Yeaststrains with this mutation show no growth defects or sensitivityto the replication inhibitor HU, suggesting that this PCNA variant issufficient for replication. However, these cells are UV-immutableindicating defective TLS. In vitro pol30–113 is an effective (albeitless than WT) processivity clamp for Pol �, but not for Pol � [23].

In conclusion, this study shows that pol � can function in TLSin vivo despite the absence of its CTD, which serves as a platform forbinding to Pol31/Pol32. The necessity of Pol32 in this mutant strainhighlights the importance of pol � integrity in TLS since Pol32 isrequired even when not a member of pol �. Furthermore, we haveshown a novel inhibitory effect of the ATPase Mgs1 specifically onthe four-subunit pol �. Both pol � and pol � have Pol32 as a subunit,but use it for somewhat different transactions. It is possible thatMgs1 can directly compete with Pol32 bound to pol � for bindingto PCNA and decrease induced mutagenesis, but does not competewith Pol32 of pol �.

Conflict of interest statement

The authors declare that there are no conflicts of interest.


Funding

This work was supported by NCI Grant CA129925 (Y.I.P.), NIGMGrant 1R01GM101167 (T.H.T), and Graduate Assistance in Areas of


ING ModelD

A Repa

Naam

A

vtRm

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[



ational Need (GAANN) fellowship #15384 in Structural Biologynd Biophysics (H.M.S.). Funding sources had no role in collection,nalysis, or interpretation of data; study design or writing of theanuscript.

cknowledgments

We would like to thank Dr. Helle Ulrich who graciously pro-ided us with several Mgs1 vectors, including YIp128-GAL-MGS1hat was used in this study. We thank Dr. Polina Shcherbakova forEV3 and REV1 expression plasmids and for critical reading of theanuscript and valuable suggestions.

eferences

[1] E.C. Friedberg, G.C. Walker, W. Siede, R.D. Wood, R.A. Schultz, T. Ellenberger,DNA Repair and Mutagenesis, 2nd ed., ASM Press, 2006.

[2] S. Boiteux, S. Jinks-Robertson, DNA repair mechanisms and the bypass of DNAdamage in Saccharomyces cerevisiae, Genetics 193 (2013) 1025–1064.

[3] E.C. Friedberg, Suffering in silence: the tolerance of DNA damage, Nat. Rev. Mol.Cell Biol. 6 (2005) 943–953.

[4] L.S. Waters, B.K. Minesinger, M.E. Wiltrout, S. D’Souza, R.V. Woodruff, G.C.Walker, Eukaryotic translesion polymerases and their roles and regulation inDNA damage tolerance, Microbiol. Mol. Biol. Rev. 73 (2009) 134–154.

[5] J.E. Sale, Translesion DNA synthesis and mutagenesis in eukaryotes, Cold SpringHarb. Perspect. Biol. 5 (2013) a012708.

[6] A.R. Lehmann, R.P. Fuchs, Gaps and forks in DNA replication: rediscovering oldmodels, DNA Repair (Amst.) 5 (2006) 1495–1498.

[7] H. Ohmori, E.C. Friedberg, R.P.P. Fuchs, M.F. Goodman, F. Hanaoka, D. Hinkle,T.A. Kunkel, C.W. Lawrence, Z. Livneh, T. Nohmi, L. Prakash, S. Prakash, T. Todo,G.C. Walker, Z. Wang, R. Woodgate, The Y-family of DNA polymerases, Mol. Cell8 (2001) 7–8.

[8] V. Pages, R.P. Fuchs, How DNA lesions are turned into mutations within cells?Oncogene 21 (2002) 8957–8966.

[9] Y.I. Pavlov, P.V. Shcherbakova, I.B. Rogozin, Roles of DNA polymerases in repli-cation, repair, and recombination, Int. Rev. Cytol. 255 (2006) 41–132.

10] W. Yang, Portraits of a Y-family DNA polymerase, FEBS Lett. 579 (2005)868–872.

11] S. Prakash, R.E. Johnson, L. Prakash, Eukaryotic translesion synthesis DNA poly-merases: specificity of structure and function, Annu. Rev. Biochem. 74 (2005)317–353.

12] Z. Livneh, O. Ziv, S. Shachar, Multiple two-polymerase mechanisms in mam-malian translesion DNA synthesis, Cell Cycle 9 (2010) 729–735.

13] G.N. Gan, J.P. Wittschieben, B.O. Wittschieben, R.D. Wood, DNA polymerase zeta(pol zeta) in higher eukaryotes, Cell Res. 18 (2008) 174–183.

14] J.E. Stone, D. Kumar, S.K. Binz, A. Inase, S. Iwai, A. Chabes, P.M. Burgers, T.A.Kunkel, Lesion bypass by S. cerevisiae Pol zeta alone, DNA Repair (Amst.) 10(2011) 826–834.

15] H.D. Ulrich, How to activate a damage-tolerant polymerase: consequences ofPCNA modifications by ubiquitin and SUMO, Cell Cycle 3 (2004) 15–18.

16] V. Pages, A. Bresson, N. Acharya, S. Prakash, R.P. Fuchs, L. Prakash, Require-ment of Rad5 for DNA polymerase zeta-dependent translesion synthesis inSaccharomyces cerevisiae, Genetics 180 (2008) 73–82.

17] S. Prakash, L. Prakash, Translesion DNA synthesis in eukaryotes: a one- or two-polymerase affair, Genes Dev. 16 (2002) 1872–1883.

18] P. Stelter, H.D. Ulrich, Control of spontaneous and damage-induced mutagen-esis by SUMO and ubiquitin conjugation, Nature 425 (2003) 188–191.

19] H.D. Ulrich, Deubiquitinating PCNA: a downside to DNA damage tolerance, Nat.Cell Biol. 8 (2006) 303–305.

20] T.T. Huang, A.D. D’Andrea, Regulation of DNA repair by ubiquitylation, Nat. Rev.Mol. Cell Biol. 7 (2006) 323–334.

21] C.W. Lawrence, V.M. Maher, Mutagenesis in eukaryotes dependent on DNApolymerase zeta and Rev1p, Philos. Trans. R. Soc. Lond. B: Biol. Sci. 356 (2001)41–46.

22] X. Zhong, P. Garg, C.M. Stith, S.A. Nick McElhinny, G.E. Kissling, P.M. Burgers,T.A. Kunkel, The fidelity of DNA synthesis by yeast DNA polymerase zeta aloneand with accessory proteins, Nucleic Acids Res. 34 (2006) 4731–4742.

23] M.R. Northam, P. Garg, D.M. Baitin, P.M.J. Burgers, P.V. Shcherbakova, Anovel function of DNA polymerase � regulated by PCNA, EMBO J. 25 (2006)4316–4325.

24] M.R. Northam, H.A. Robinson, O.V. Kochenova, P.V. Shcherbakova, Participationof DNA polymerase zeta in replication of undamaged DNA in Saccharomycescerevisiae, Genetics 184 (2010) 27–42.

25] M.R. Northam, E.A. Moore, T.M. Mertz, S.K. Binz, C.M. Stith, E.I. Stepchenkova,K.L. Wendt, P.M. Burgers, P.V. Shcherbakova, DNA polymerases zeta and Rev1



mediate error-prone bypass of non-B DNA structures, Nucleic Acids Res. 42(2014) 290–306.

26] P.A. Auerbach, B. Demple, Roles of Rev1, Pol zeta, Pol32 and Pol eta in the bypassof chromosomal abasic sites in Saccharomyces cerevisiae, Mutagenesis 25 (2010)63–69.

[

[

PRESSir xxx (2014) xxx–xxx 11

27] J.P. Wittschieben, S.C. Reshmi, S.M. Gollin, R.D. Wood, Loss of DNA polymerasezeta causes chromosomal instability in mammalian cells, Cancer Res. 66 (2006)134–142.

28] L.A. Loeb, R.J. Monnat Jr., DNA polymerases and human disease, Nat. Rev. Genet.9 (2008) 594–604.

29] L.J. Stallons, W.G. McGregor, Translesion synthesis polymerases in the pre-vention and promotion of carcinogenesis, J. Nucleic Acids 2010 (2010), pii:643857.

30] X. Lin, J. Trang, T. Okuda, S.B. Howell, DNA polymerase zeta accounts for thereduced cytotoxicity and enhanced mutagenicity of cisplatin in human coloncarcinoma cells that have lost DNA mismatch repair, Clin. Cancer Res. 12 (2006)563–568.

31] J.R. Nelson, C.W. Lawrence, D.C. Hinkle, Thymine–thymine dimer bypass byyeast DNA polymerase �, Science 272 (1996) 1646–1649.

32] A.G. Baranovskiy, A.G. Lada, H.M. Siebler, Y. Zhang, Y.I. Pavlov, T.H. Tahirov, DNApolymerase � and � switch by sharing accessory subunits of DNA polymerase�, J. Biol. Chem. 287 (2012) 17281–17287.

33] Y.S. Lee, M.T. Gregory, W. Yang, Human Pol zeta purified with accessory sub-units is active in translesion DNA synthesis and complements Pol eta in cisplatinbypass, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 2954–2959.

34] R.E. Johnson, L. Prakash, S. Prakash, Pol31 and Pol32 subunits of yeast DNApolymerase delta are also essential subunits of DNA polymerase zeta, Proc.Natl. Acad. Sci. U.S.A. 109 (2012) 12455–12460.

35] A.V. Makarova, J.L. Stodola, P.M. Burgers, A four-subunit DNA polymerasezeta complex containing Pol delta accessory subunits is essential for PCNA-mediated mutagenesis, Nucleic Acids Res. (2012) 1–9.

36] L.D. Langston, M. O’Donnell, Subunit sharing among high- and low-fidelity DNApolymerases, Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 12268–12269.

37] A.G. Baranovskiy, N.D. Babayeva, V.G. Liston, I.B. Rogozin, E.V. Koonin, Y.I.Pavlov, D.G. Vassylyev, T.H. Tahirov, X-ray structure of the complex of regula-tory subunits of human DNA polymerase delta, Cell Cycle 7 (2008) 3026–3036.

38] T.H. Tahirov, Structure and structure and function of eukaryotic DNA poly-merase �, Subcell. Biochem. 62 (2012) 217–236.

39] Y. Gomez-Llorente, R. Malik, R. Jain, J.R. Choudhury, R.E. Johnson, L. Prakash, S.Prakash, I. Ubarretxena-Belandia, A.K. Aggarwal, The architecture of yeast DNApolymerase zeta, Cell Rep. 5 (2013) 79–86.

40] A. Daraba, V.K. Gali, M. Halmai, L. Haracska, I. Unk, Def1 promotes the degra-dation of Pol3 for polymerase exchange to occur during DNA-damage-inducedmutagenesis in Saccharomyces cerevisiae, PLoS Biol. 12 (2014) e1001771.

41] S. Klinge, R. Nunez-Ramirez, O. Llorca, L. Pellegrini, 3D architecture of DNAPol alpha reveals the functional core of multi-subunit replicative polymerases,EMBO J. 28 (2009) 1978–1987.

42] D.J. Netz, C.M. Stith, M. Stumpfig, G. Kopf, D. Vogel, H.M. Genau, J.L. Stodola,R. Lill, P.M. Burgers, A.J. Pierik, Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes, Nat. Chem. Biol. 8 (2012)125–132.

43] I.S. Waisertreiger, V.G. Liston, M.R. Menezes, H.M. Kim, K.S. Lobachev, E.I.Stepchenkova, T.H. Tahirov, I.B. Rogozin, Y.I. Pavlov, Modulation of mutagene-sis in eukaryotes by DNA replication fork dynamics and quality of nucleotidepools, Environ. Mol. Mutagen. 53 (2012) 699–724.

44] I. Saugar, J.L. Parker, S. Zhao, H.D. Ulrich, The genome maintenance factor Mgs1is targeted to sites of replication stress by ubiquitylated PCNA, Nucleic AcidsRes. 40 (2012) 245–257.

45] Y.I. Pavlov, P.V. Shcherbakova, T.A. Kunkel, In vivo consequences of putativeactive site mutations in yeast DNA polymerases �, �, �, and �, Genetics 159(2001) 47–64.

46] P.M. Burgers, Overexpression of multisubunit replication factors in yeast, Meth-ods 18 (1999) 349–355.

47] P. Garg, C.M. Stith, J. Majka, P.M. Burgers, Proliferating cell nuclear antigenpromotes translesion synthesis by DNA polymerase zeta, J. Biol. Chem. 280(2005) 23446–23450.

48] S.G. Kozmin, Y.I. Pavlov, T.A. Kunkel, E. Sage, Roles of Saccharomyces cerevisiaeDNA polymerases Pol� and Pol� in response to irradiation by simulated sun-light, Nucleic Acids Res. 31 (2003) 4541–4552.

49] T.H. Tahirov, K.S. Makarova, I.B. Rogozin, Y.I. Pavlov, E.V. Koonin, Evolutionof DNA polymerases: an inactivated polymerase-exonuclease module in Polepsilon and a chimeric origin of eukaryotic polymerases from two classes ofarchaeal ancestors, Biol. Direct 4 (2009) 10.

50] W.L. Whelan, E. Gocke, T.R. Manney, The CAN1 locus of Saccharomyces cere-visiae: fine-structure analysis and forward mutation rates, Genetics 91 (1979)35–51.

51] K.J. Gerik, X. Li, A. Pautz, P.M. Burgers, Characterization of the two small subunitsof Saccharomyces cerevisiae DNA polymerase delta, J. Biol. Chem. 273 (1998)19747–19755.

52] M.E. Huang, A. de Calignon, A. Nicolas, F. Galibert, POL32, a subunit of theSaccharomyces cerevisiae DNA polymerase delta, defines a link between DNAreplication and the mutagenic bypass repair pathway, Curr. Genet. 38 (2000)178–187.

53] N. Acharya, L. Haracska, R.E. Johnson, I. Unk, S. Prakash, L. Prakash, Complexformation of yeast Rev1 and Rev7 proteins: a novel role for the polymerase-associated domain, Mol. Cell. Biol. 25 (2005) 9734–9740.


54] T. Hishida, T. Ohya, Y. Kubota, Y. Kamada, H. Shinagawa, Functional and physicalinteraction of yeast Mgs1 with PCNA: impact on RAD6-dependent DNA damagetolerance, Mol. Cell. Biol. 26 (2006) 5509–5517.

55] L. Giot, R. Chanet, M. Simon, C. Facca, G. Faye, Involvement of the yeast DNApolymerase � in DNA repair in vivo, Genetics 146 (1997) 1239–1251.


http://refhub.elsevier.com/S1568-7864(14)00135-9/sbref0005





















































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































ING ModelD

1 A Repa

[

[

[

[

[

[

[

[


2 H.M. Siebler et al. / DN

56] T.A. Kunkel, P.M. Burgers, Dividing the workload at a eukaryotic replicationfork, Trends Cell Biol. 18 (2008) 521–527.

57] V. Pages, R.E. Johnson, L. Prakash, S. Prakash, Mutational specificity and geneticcontrol of replicative bypass of an abasic site in yeast, Proc. Natl. Acad. Sci. U.S.A.105 (2008) 1170–1175.

58] Y.I. Pavlov, P.V. Shcherbakova, DNA polymerases at the eukaryotic fork-20 yearslater, Mutat. Res. 685 (2010) 45–53.

59] S. Kikuchi, K. Hara, T. Shimizu, M. Sato, H. Hashimoto, Structural basis of recruit-ment of DNA polymerase zeta by interaction between REV1 and REV7 proteins,



J. Biol. Chem. 287 (2012) 33847–33852.60] N. Acharya, R.E. Johnson, V. Pages, L. Prakash, S. Prakash, Yeast Rev1 pro-

tein promotes complex formation of DNA polymerase zeta with Pol32subunit of DNA polymerase delta, Proc. Natl. Acad. Sci. U.S.A. 106 (2009)9631–9636.

[

PRESSir xxx (2014) xxx–xxx

61] T. Hishida, H. Iwasaki, T. Ohno, T. Morishita, H. Shinagawa, A yeast gene, MGS1,encoding a DNA-dependent AAA(+) ATPase is required to maintain genomestability, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 8283–8289.

62] H. Nomura, A. Yoshimura, T. Edo, S. Kanno, S. Tada, M. Seki, A. Yasui, T.Enomoto, WRNIP1 accumulates at laser light irradiated sites rapidly via itsubiquitin-binding zinc finger domain and independently from its ATPasedomain, Biochem. Biophys. Res. Commun. 417 (2012) 1145–1150.

63] T. Tsurimoto, A. Shinozaki, M. Yano, M. Seki, T. Enomoto, Human Werner heli-case interacting protein 1 (Wrnip1) functions as a novel modulator for DNA


polymerase �, Genes Cells 10 (2005) 13–22.64] D. Branzei, M. Seki, F. Onoda, T. Enomoto, The product of Saccharomyces

cerevisiae WHIP/MGS1, a gene related to replication factor C genes, interactsfunctionally with DNA polymerase delta, Mol. Genet. Genomics 268 (2002)371–386.









































































































































































































































































































g model article in pressmicrobiology.ucdavis.edu/kowalczykowski/pdf_files/siebler et al, 201… ·...

Documents