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DNA Repair 12 (2013) 878– 889

Contents lists available at ScienceDirect

DNA Repair

j ourna l ho me p age : www.elsev ier .com/ locate /dnarepai r

he choice of nucleotide inserted opposite abasic sites formed withinhromosomal DNA reveals the polymerase activities participating inranslesion DNA synthesis

in Chan, Michael A. Resnick, Dmitry A. Gordenin ∗

hromosome Stability Section, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, 111.W. Alexander Drive, Research Triangle Park, NC 27709, USA

r t i c l e i n f o

rticle history:eceived 21 June 2013eceived in revised form 19 July 2013ccepted 20 July 2013vailable online 26 August 2013

eywords:basic siteranslesion DNA synthesisPOBECingle-strand DNAeoxycytidyltransferase

a b s t r a c t

Abasic sites in genomic DNA can be a significant source of mutagenesis in biological systems, includinghuman cancers. Such mutagenesis requires translesion DNA synthesis (TLS) bypass of the abasic siteby specialized DNA polymerases. The abasic site bypass specificity of TLS proteins had been studiedby multiple means in vivo and in vitro, although the generality of the conclusions reached have beenuncertain. Here, we introduce a set of yeast reporter strains for investigating the in vivo specificity ofabasic site bypass at numerous random positions within chromosomal DNA. When shifted to 37 ◦C, thesestrains underwent telomere uncapping and resection that exposed reporter genes within a long 3′ ssDNAoverhang. Human APOBEC3G cytosine deaminase was expressed to create uracils in ssDNA, which wereexcised by uracil-DNA N-glycosylase. During repair synthesis, error-prone TLS bypassed the resultingabasic sites. Because of APOBEC3G’s strict motif specificity and the restriction of abasic site formation toonly one DNA strand, this system provides complete information about the location of abasic sites that

led to mutations. We recapitulated previous findings on the roles of REV1 and REV3. Further, we foundthat sequence context can strongly influence the relative frequency of A or C insertion. We also found thatdeletion of Pol32, a non-essential common subunit of Pols � and �, resulted in residual low-frequencyC insertion dependent on Rev1 catalysis. We summarize our results in a detailed model of the interplaybetween TLS components leading to error-prone bypass of abasic sites. Our results underscore the utilityof this system for studying TLS bypass of many types of lesions within genomic DNA.

. Introduction

DNA is under constant threat of damage by both endogenous andnvironmental agents [1]. Damage to the nitrogenous bases thatesult in miscoding can be particularly deleterious if left unrepairedefore a subsequent round of replication, as this can result in muta-ion fixation [1]. A principal means of correcting various adductesions is by base excision repair (BER), which is initiated when

DNA N-glycosylase specifically recognizes its cognate adductedase substrate and excises it to generate an abasic site [2]. Aba-ic sites also are generated when glycosylases erroneously excise

ormal bases [3] or by spontaneous breakage of the N-glycosidicond, especially in adducted nucleotides where this bond is desta-ilized [4]. Usually, short- or long-patch BER replaces the damaged

∗ Corresponding author at: 111 T.W. Alexander Drive, P.O. Box 12233, Researchriangle Park, NC 27709, USA. Tel.: +1 919 541 5190; fax: +1 919 541 7593.

E-mail addresses: chank2@niehs.nih.gov (K. Chan), resnick@niehs.nih.govM.A. Resnick), gordenin@niehs.nih.gov (D.A. Gordenin).

568-7864/$ – see front matter. Published by Elsevier B.V.ttp://dx.doi.org/10.1016/j.dnarep.2013.07.008

Published by Elsevier B.V.

nucleotide (along with some neighboring residues, in the lattercase), using the intact complementary strand as a template [2].

Abasic sites are thought to be one of the most common lesionswithin DNA, under steady state, unstressed conditions [5]. Whileone would expect that the majority of such lesions should berepaired correctly by BER, abasic sites formed within single-strandDNA (ssDNA) likely would not be subject to such error-free repair.This is because the complementary strand to template repairsynthesis is, by definition, absent. Moreover, it would be coun-terproductive to attempt BER on an abasic site within ssDNA ofa replication fork, as doing so would risk strand breakage and forkcollapse [6]. Similarly, BER would be unavailable for repairing aba-sic sites formed by strand resection from double-strand breaksor within subtelomeric ssDNA following telomere uncapping. Inthe latter scenario, we previously observed clusters of multiplebase substitutions originating from abasic sites on the 3′ ssDNA

overhang strand [7]. More recently, Neuberger and colleaguesexpressed hyperactive cytosine deaminases in yeast and found sim-ilar mutation showers, which were associated with breakpoints andthought to result from damage to ssDNA at resected double strand

K. Chan et al. / DNA Repair 12 (2013) 878– 889 879

Table 1List of yeast strains used in this study.

Strain name(s) Genotype Plasmid

yKC066, yKC067 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 lys2::CAN1-URA3-ADE2 (in sub-telomere 5L)a pCM252-A3GyKC068, yKC069 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 lys2::CAN1-URA3-ADE2 (in sub-telomere 5L)a pCM252yKC213, yKC214 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 rad30::HygR lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252-A3GyKC217, yKC218 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 rad30::HygR lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252yKC131, yKC135 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 rev3::NatR lys2::CAN1-URA3-ADE2 (in sub-telomere 5L)a pCM252-A3GyKC133, yKC137 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 rev3::NatR lys2::CAN1-URA3-ADE2 (in sub-telomere 5L)a pCM252yKC205, yKC206 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 rev1::HygR lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252-A3GyKC209, yKC210 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 rev1::HygR lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252yKC385 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 rev1-AA lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252-A3GyKC387, yKC388 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 rev1-AA lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252yKC299, yKC230 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 rad30::HygR rev3::NatR lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252-A3GyKC233, yKC234 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 rad30::HygR rev3::NatR lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252yKC245, yKC246 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 rad30::KanMX4 rev1::HygR lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252-A3GyKC249, yKC250 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 rad30::KanMX4 rev1::HygR lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252yKC261, yKC262 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 pol32::HygR lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252-A3GyKC265 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 pol32::HygR lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252yKC399 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 pol32::HygR rev1-AA lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252-A3G

R rev1

s origi

biam

talsi(cctAp[ab[

irsffseurPtiie[ltfroPT

s

yKC400, yKC402 MAT ̨ his7-2 leu2-3,112 trp1-289 cdc13-1 pol32::Hyg

a The WT and rev3 strains were described previously in [7]. All other yeast strain

reaks [8]. Such formations as observed by both groups are rem-niscent of similar clusters of multiple point mutations identifiedmong various human cancers, also likely to have originated fromany abasic sites within long stretches of ssDNA [9–11].When confronted with abasic sites in a DNA configuration

hat is not amenable to BER, the cell resorts to using mech-nisms of DNA damage tolerance that result in bypass of theesion without repairing it (reviewed recently in [6,12]). Oneuch mechanism is translesion DNA synthesis (TLS) by special-zed DNA polymerases, which can be error-prone, i.e. mutagenicreviewed recently in [12–14]). This is because TLS polymerasesan contain relatively large active sites that accommodate non-anonical bases (e.g., [15,16]) and lack the proofreading activity ofhe higher fidelity replicative polymerases (reviewed in [17,18]).s a result, TLS polymerases are considerably more error-rone, even when copying undamaged DNA templates (e.g.,19,20]). Thus, cells have evolved these low fidelity polymerasess a means to bypass genomic lesions that would otherwiselock replication, but at the cost of possible mutation fixation12–14].

Conserved proteins that take part in TLS have been identifiedn many organisms, including budding yeast [21–24]. Yeast has aepertoire of three specialized TLS polymerases whose functionalpecificity in bypassing abasic sites has been investigated by theollowing approaches: in vitro TLS assays (e.g., [25–27]); trans-ecting cells with plasmids carrying a single, engineered abasicite [28–30]; expressing mutator DNA glycosylases that generatexcess abasic sites in BER-deficient strains [31,32]; and analyzingnselected abasic site-associated TLS events within a frameshifteversion reporter, in excision repair defective backgrounds [33].ol �, which catalyzes error-free bypass of UV-induced cyclobu-ane pyrimidine dimers, is encoded by the RAD30 gene [34]. Pol �s proficient at extending from various mismatched termini dur-ng TLS in vitro (e.g., [27,35,36]) and includes a catalytic subunitncoded by REV3 along with an accessory subunit encoded by REV737]. There is also evidence that Pol �, acting alone, can bypass someesions in vitro [37,38]. REV1 encodes a protein with deoxycytidyl-ransferase activity [26] and provides a structural function crucialor TLS bypass of certain lesions (e.g., [39–41]). Additionally, theeplicative polymerase � has been implicated in the insertion of Apposite abasic sites [27]. A non-essential accessory subunit of both

ol � and Pol �, encoded by POL32 [42–44], is necessary for efficientLS bypass of various lesions, including abasic sites [29,30].

Previously, we used a subtelomeric ssDNA mutagenesis reporterystem to confirm that the mutagenic bypass of abasic sites

-AA lys2::CAN1-URA3-ADE2 (in sub-telomere 5L) pCM252

nate from this study.

(resulting from Ung1-catalyzed excision of uracils [45] that wereformed by cytosine deamination) within chromosomal DNArequired the TLS activity of Pol � [7]. In contrast, when uracilswere left intact in ung1� cells, mutagenesis was completely inde-pendent of TLS, as deletion of REV3 did not affect the frequencyof gene inactivation at all in this background [7]. Thus, the TLSdependence in cells with Ung1 argues that mutagenesis due toresidual amounts of unexcised uracils was at most, a minor factor.Here, we apply the subtelomeric ssDNA mutagenesis reportersystem to investigate the roles of TLS proteins in the error-pronebypass of randomly generated abasic sites within chromosomalDNA. We found that similar to human cancers [9–11], A and C tendto be incorporated opposite abasic sites at similar frequencies.Based on our results, we infer the respective roles and relativecontributions of the various TLS proteins toward the error-pronebypass of abasic sites within genomic DNA.

2. Materials and methods

2.1. Yeast strains

Yeast strains used in this study are listed in Table 1. All are iso-genic to CG379 [46] with the following common markers: MAT˛his7-2 leu2-3,112 trp1-289. WT and rev3 subtelomeric reporterstrains bearing tetracycline regulatable APOBEC3G plasmids weredescribed previously [7]. Other TLS gene deletion strains were con-structed by one step gene replacement [47]. The rev1-AA allele (withD647A and E648A amino acid substitutions [27]) was constructedby the delitto perfetto approach [48]. Strain constructions were veri-fied by diagnostic replica plating, PCR, and sequencing (for rev1-AA).Strains were maintained on TRP dropout media to select for plasmidretention.

2.2. Determination of CAN1 inactivation frequency

For each combination of genotype and plasmid, multiple inde-pendent colonies each were inoculated into 5 mL of YPDA liquid (1%yeast extract, 2% peptone, 2% dextrose, supplemented with 0.01%adenine sulfate, filter-sterilized) and grown at 23 ◦C for 2 days.Then, 500 �L of each culture was added to 4.5 mL of fresh YPDAwith 10 �g/mL doxycycline hyclate [49] (Sigma–Aldrich, St. Louis,

MO) and shifted to 37 ◦C for 6 h. Cells were counted and G2 arrestwas monitored by examining cell morphology. Cells were washedonce in water. 500 cells were plated, in triplicate, onto syntheticcomplete to determine plating efficiency. An appropriate dilution

880 K. Chan et al. / DNA Repair 12 (2013) 878– 889

Fig. 1. Genetic construct and experimental design to assess mutagenesis arising from random abasic sites within subtelomeric ssDNA. (A) Each reporter strain bears thecdc13-1 mutation and three reporter genes (ADE2, URA3, and CAN1), near a de novo telomere on the left arm of Chromosome V. (B) Temperature shift results in dissociationof the protective proteinaceous cap of the telomere and enzymatic 5′ to 3′ resection, generating a long 3′ overhang of ssDNA. Simultaneously, APOBEC3G cytosine deaminasee ment

w acils.

C trate fs

fwtfgf

2

tat

xpression is driven by adding doxycycline. c(ADE2) and c(CAN1) denote the compleithin the ssDNA overhang. Ung1 generates abasic sites by excising the resulting ur

opposite the abasic sites. The restored duplex, containing abasic site(s), is a substrands.

or each strain was plated, in triplicate, onto ARG dropout platesith 60 mg/mL canavanine sulfate and 20 mg/mL adenine sulfate

o select for mutants. Plates were counted after incubation at 23 ◦Cor 5 days. Canavanine selection plates were replica plated ontolycerol plates to eliminate cultures started by petite colonies fromurther consideration.

.3. Collection and sequencing of CAN1 inactivation mutants

Cultures derived from independent colonies were started andemperature shifted as described in Section 2.2. From each culture,n appropriate dilution was plated onto a single canavanine selec-ion plate and incubated for 5 days at 23 ◦C. A single colony, located

of the two genes. (C) APOBEC3G deaminates the 3′ cytosines of CC motifs at random(D) When restored to permissive temperature, error-prone TLS usually inserts A oror base excision repair. (E) After base excision repair, mutations are fixed on both

closest to a fixed point on each plate, was streaked onto YPDA.A single colony from each streak was patched onto YPDA, grownfor 2 days at 23 ◦C, and replica plated onto canavanine media toverify loss of CAN1 function, and onto glycerol media to verify nor-mal aerobic respiration capacity. Genomic DNA was prepared fromeach mutant isolate using a QIAcube robot (QIAGEN, Valencia, CA),according to the manufacturer’s DNeasy protocol. Using standardmethods, CAN1 was PCR amplified and sequenced (primers arelisted in Supplementary Table 1), and mutations were identified,

as described previously [7].

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.dnarep.2013.07.008.

K. Chan et al. / DNA Repair

Fig. 2. CAN1 inactivation frequencies in cdc13-1 reporter strains expressingAPOBEC3G cytosine deaminase during temperature shift-induced generation of sub-telomeric ssDNA. Each data point represents an independent replicate derived froma single colony, numbers indicate median values, and asterisks denote significantdifference (P < 0.05) compared to WT by the Kolmogorov–Smirnov test. Canavaninese

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3

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cesrCedrtwretoowcgbc

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election plates were counted after 5 days of growth at 23 ◦C following mutagenicxposure. See Section 2 for details of experimental procedures.

.4. Statistical analyses

Prism 6 software (GraphPad Software, LaJolla, CA) was used toarry out statistical tests on the data. The Kolmogorov–Smirnovest was used for pairwise comparisons (between TLS genotypes) ofAN1 inactivation frequency and plating efficiency. The two-tailed-by-2 Fisher’s exact test or the Chi-square test was used to com-are relative proportions of different mutation types between TLSenotypes, or between APOBEC3G vs. empty vector in the same TLSenotype.

. Results

.1. A model system to assess the roles of TLS proteins in theutagenic bypass of abasic sites within chromosomal DNA

The experimental design is summarized in Fig. 1. When reporterells are shifted from 23 to 37 ◦C, telomere uncapping and ensuingnzymatic resection produce long 3′ ssDNA overhangs [50]. Theequences that either code for, or are complementary to, threeeporter genes are located within the left subtelomeric region ofhromosome V. We expressed human APOBEC3G [51,52] to gen-rate abasic sites in the subtelomeric ssDNA overhang. APOBEC3Geaminates the 3′ C within CC motifs [53] of the ssDNA reporteregion at random, but does not react with duplex DNA [54]. Ung1hen excises the resulting uracils to generate abasic sites [45],hich are bypassed by error-prone TLS during re-synthesis of the

esected strand, when cells are returned to 23 ◦C. This series ofvents results in mutations that are identified by plating on mediahat selects cells with inactivated CAN1 [55]. A unique strengthf this experimental design is that it enables the characterizationf mutagenic bypass at endogenously generated abasic sites, at aell-defined sequence motif, in numerous random locations within

hromosomal DNA. This approach should yield results of broaderenerality than other methods, e.g. surveying TLS opposite plasmid-orne single, engineered abasic sites that are introduced into theell by transfection [28–30].

To characterize the roles of proteins involved in TLS, werst determined the frequency of CAN1 inactivation among cellsxpressing APOBEC3G during temperature shift, as a means to esti-ate the frequency of mutagenic TLS at abasic sites (see Fig. 2). Data

12 (2013) 878– 889 881

on plating efficiencies and CAN1 inactivation frequencies of emptyvector controls are in Supplementary Figures 1 and 2, respectively.Consistent with in vivo studies based on transfection of plasmidsbearing a single, designed abasic site [29,30], we found that REV3and REV1 both were necessary for the vast majority of mutagenicTLS, while deletion of RAD30 did not affect the frequency of TLSsignificantly compared to WT. In contrast to cells deleted for REV1,we found that cells lacking only Rev1 catalytic deoxycytidyltrans-ferase activity [27] (i.e. rev1-AA cells) exhibited a gene inactivationfrequency that was indistinguishable from WT, supporting priorobservations that only the structural function of Rev1 is requiredfor WT levels of TLS at a model abasic site [30]. Deletion of POL32also resulted in a significant decrease in frequency of TLS whencompared to WT, consistent with previous reports [29,30]. Finally,deletion of POL32 in the rev1-AA background resulted in a further4.4-fold decrease in frequency of TLS when compared to the pol32single mutant (P = 0.0043 by Kolmogorov–Smirnov test), suggest-ing that the majority of low frequency, residual TLS in pol32 cellsinvolved Rev1 catalytic activity.

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.dnarep.2013.07.008.

3.2. Sequence context can influence the choice of nucleotideinserted opposite abasic sites in WT cells

To glean further insight into the molecular mechanisms under-lying mutagenic abasic site bypass, we sequenced the CAN1 ORFfrom independent mutant isolates. Starting with cells WT for TLS,we sequenced 84 isolates, which yielded 91 mutations in totalwithin CAN1. Detailed mutation data for all genotypes, eitherexpressing APOBEC3G or bearing empty vector, are in Supplemen-tary Tables 2 and 3, respectively. 74 of the mutations occurred atthe 3′ C of CC motifs, i.e. at the cognate motif of APOBEC3G, andthus were likely to have originated from abasic sites. 41 were CCto CT transitions (i.e., insertion of A opposite the abasic site), while33 were CC to CG transversions (insertion of C). To obtain the fre-quency of a given class of mutations, we multiplied the overall CAN1inactivation frequency (1.36 × 10−3 for WT) by the ratio of the num-ber of mutations of that class to the total number of mutations (seeFig. 3A).

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.dnarep.2013.07.008.

While mutations were found at 19 CC sites spread across theCAN1 reporter, we noticed that some positions contained consid-erably more mutations than others (Fig. 4A). Specifically, 32 out of74 mutations occurred in just two positions, C530 and C718, whereboth CC to CT and CC to CG substitutions were observed (see alsoSupplementary Table 4). The C to G allele at position 530 results ina tryptophan to serine substitution, while the C to T allele resultsin an amber stop codon. Both the C to G and C to T alleles at posi-tion 718 yield glycine to arginine substitutions. (Since the codingstrand for CAN1 is complementary to the ssDNA overhang left byresection from the uncapped telomere, the actual coding changesare complementary to the mutations occurring at CC motifs inthe ssDNA overhang.) All four alleles (C530G, C530T, C718G, andC718T) were clearly canavanine resistant. Interestingly, these pos-itions differed for the relative proportion of CC to CT vs. CC to CG(P = 0.0006 by Fisher’s exact test; see also Fig. 4A). There was a clearpreference for insertion of C across an abasic site at position530 versus a preference for insertion of A across an abasic site

at position 718, suggesting a genuine bias in favor of one typeof TLS transaction over the other. We propose that the localsequence context at these two positions can influence the choice ofnucleotide inserted opposite the abasic site. However, it is not clear

882 K. Chan et al. / DNA Repair 12 (2013) 878– 889

Fig. 3. Frequencies of various types of mutations. Numbers above each bar indicate the number of independent mutant isolates within each category. (A) Mutations aregrouped as CC to CT, CC to CG, and all others. Note that significant proportions of mutations occurred at the APOBEC3G cognate motif, namely CC. (B) Two mutational hotspots,C530 and C718 are shown for WT, rad30, and rev1-AA, three genetic backgrounds that are proficient for TLS bypass opposite abasic sites. Abasic sites at C530 favored TLSi stitutC

wt(

f2

wHsitr(tp(nab

3T

t

nsertion of C. In contrast at C718, insertion of A was favored. (C) Frequencies of sub718G substitutions in pol32 was unique among TLS deficient genotypes.

hich differences in sequence could be influencing this choice, ashere are many differences in the respective flanking sequencessee Section 4).

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.dnarep.013.07.008.

We also noticed that the highest number of CC to CT transitionsas observed in position 857, which creates an amber stop codon.owever the C857G allele, which would result in a tryptophan to

erine substitution, was never isolated as an inactivating mutationn any of numerous studies with published CAN1 mutation spec-ra [53,56–60]. Similarly, we have never observed a C857G alleleesulting in gene inactivation among existing datasets in our group[10,61,62] and unpublished observations). As it is quite possiblehat the C857G allele does not inactivate CAN1, we do not considerosition 857 to be a site where any preference for insertion of Ainstead of C) can be inferred. Nonetheless, the sharp contrast inucleotide insertion preferences between positions 530 and 718rgues that not all abasic sites are processed in the same mannery the TLS machinery.

.3. Rad30 (Pol �) does not play a significant role in mutagenic

LS opposite abasic sites

Consistent with previous studies [29,30], we found that dele-ion of RAD30 did not alter the frequency of mutagenic TLS opposite

ions at C530 and C718 for TLS deficient backgrounds. The prevalence of C530G and

abasic sites significantly, when compared to WT (see Fig. 2). If Rad30does not play any significant role in TLS opposite abasic sites, thenthe mutation spectrum of rad30 cells also should be essentiallythe same as that of WT. We sequenced 48 independent isolatesfrom rad30 cultures treated with APOBEC3G, to obtain 52 muta-tions. Similar to WT, the large majority of mutations (39 of 52)occurred at CC motifs and there were similar numbers of CC toCT transitions (19) and CC to CG transversions (20) (see Fig. 3A).Also similar to WT, the rad30 mutation spectrum revealed a CC toCG preference at position 530 and a CC to CT preference at position718 (P = 0.0006 when comparing these two sites by Fisher’s exacttest; see Fig. 3B and Supplementary Table 4). Application of theChi-square test to compare relative proportions of mutation typesconfirmed that there was no significant difference between WT andrad30 (see Table 2).

The only apparent possible difference between the WT andrad30 spectra was at position 806 (compare Fig. 4A to B). InWT, this site exhibited three CC to CT transitions, one CC toCG transversion, and five single base deletions of C, i.e. ninemutations in total. By contrast in rad30, we observed a lone CCto CG transversion. Yet, comparing the relative ratios of muta-tions at C806, between WT and rad30 by two-tailed Fisher’s

exact test, we obtained a P-value that was not quite statisti-cally significant (P = 0.0936). Altogether, we conclude that Rad30plays, at most, a negligible role in the bypass of abasic sites invivo.

K. Chan et al. / DNA Repair 12 (2013) 878– 889 883

Fig. 4. The mutation spectra of (A) WT and (B) rad30 strains expressing APOBEC3G during temperature shift. Position 530 exhibited a strong preference for inserting C oppositethe abasic site, resulting in frequent CC to CG transversions. Position 718 exhibited a preference for inserting A opposite the abasic site, resulting in CC to CT transitions.C rsion,r

3i

�w

TC

857 exhibited CC to CT transitions exclusively, possibly because the C857G transveespective X- and Y-axes in this figure and Figs. 5 and 6 are of the same scale.

.4. Rev3 and Rev1 are required for efficient TLS, and for thensertion of C opposite abasic sites

Deletion of either REV3 (encoding the catalytic subunit of Pol) or REV1 resulted in 66- and 51-fold decreases in TLS frequencyhen compared to WT, respectively (see Fig. 2). Dividing the CAN1

able 2omparison of proportions of mutation types between WT and each TLS mutant.

Genotype CC to CG transversions CC to CT transitio

WT 33 41

rad30 19 20

rev3 0 21

rev1 0 29

rev1-AA 0 31

rad30 rev3 0 22

rad30 rev1 0 28

pol32 20 7

pol32 rev1-AA 0 19

resulting in a tryptophan to serine amino acid change, did not inactivate CAN1. The

inactivation frequency of each mutant by that of WT, we infer thatrev3 retained 1.5% of the TLS capacity of WT, while rev1 retained

1.9% of WT capacity. This is in good agreement with the resultsof Lawrence and colleagues, who obtained values of 4.9% and 3.4%for rev3 and rev1, respectively, using a plasmid-based in vivo assay[28]. Prakash and colleagues, using a similar approach as Lawrence,

ns All other mutations Chi-square P-value vs. WT

17 n/a13 0.616419 <0.000117 <0.000114 <0.000122 <0.000119 <0.000121 0.0004

4 0.0013

8 epair

drsc

or(otWTaC3CitvcT

r[atptFpwprttc

bFRbP

3cs

adTwirrFs

Ad4Cpo(c

84 K. Chan et al. / DNA R

erived values of 8% and 41% of WT TLS capacity for rev3 and rev1,espectively [30]. Thus, the rev3 data for all three studies wereimilar, while the higher TLS capacity observed by Prakash andolleagues for rev1 was not observed in Lawrence’s system or ours.

We sequenced 40 rev3- and 46 rev1-derived isolates, each withne mutation in CAN1 and found that the mutation spectra for bothev3 and rev1 were completely devoid of CC to CG transversionssee Fig. 3A). In contrast, CC to CT transitions comprised over halff total mutations for both genotypes. The differences in propor-ion of mutation types were highly significant when compared to

T (P < 0.0001 by Chi-square test for either rev3 or rev1 vs. WT, seeable 2). Only two out of 21 independent isolates each from rev3nd rev1 bearing empty vector exhibited CC to CT transitions (noC to CG transversions were observed; see Supplementary Table). Fisher’s exact test P-values comparing the proportion of CC toT mutations in rev3 cells expressing APOBEC3G vs. empty vector

s 0.0009. The corresponding P-value for rev1 cells is 0.0001. Thus,he comparison between APOBEC3G-expressing strains to emptyector controls clearly showed the mutagenic specificity of thisytosine deaminase, even in genetic backgrounds that were grosslyLS deficient.

The lack of C insertion in our system is in good agreement withesults from Lawrence’s group [29], but less so with Prakash’s group30], who observed 31% and 10% insertion of C opposite their modelbasic site in the rev3 and rev1 backgrounds, respectively. In addi-ion, our mutation spectra exhibited a strong CC to CT hotspot atosition 718 (see Figs. 5A, 5B, and 3C). Also note that position 718ended to favor insertion of A (CC to CT transitions) in WT (seeigs. 3B, 4A, and Supplementary Table 4). Our results suggest thatosition 718 might be relatively easy to bypass, even in genotypesith severe TLS deficiency, perhaps by the action of replicativeolymerase � alone. While we cannot rule out the possibility thatesidual CC to CT transitions observed in rev3 or rev1 could be dueo small numbers of unexcised uracils, the overall agreement withhe results of Lawrence and coworkers [29] as well as Prakash andoworkers [30] is consistent with low-frequency TLS.

Finally, we found that rad30 rev3 and rad30 rev1 double mutantsehaved similarly to rev3 and rev1 single mutants, respectively (seeigs. 2, 3A, C, and Supplementary Table 4). These results suggest thatad30 truly does not play any significant role during mutagenicypass of abasic sites, not even in a backup capacity when eitherol � or Rev1 are unavailable.

.5. The structural function of Rev1 enables efficient TLS, butatalytic function is necessary for insertion of C opposite abasicites

Rev1 is thought to serve a dual role in enabling TLS. It possesses deoxycytidyltransferase activity and a structural role (indepen-ent of its enzymatic function) that facilitates the activity of otherLS proteins (reviewed in [12–14]). Since no CC to CG mutationsere found among the 46 independent rev1-derived isolates with

nactivated CAN1, we tested whether Rev1 catalytic activity wasesponsible for insertion of C opposite abasic sites. As mentioned,ev1-AA cells exhibited WT levels of overall TLS frequency (seeig. 2). This indicates that Rev1’s structural function is in itself,ufficient to support efficient TLS, in some form.

Although the overall TLS frequencies were similar between rev1-A and WT, the compositions of the mutation spectra were radicallyifferent. We sequenced 42 independent rev1-AA isolates to obtain5 mutations in CAN1. 31 mutations were CC to CT while none wereC to CG (see Fig. 3A). With the loss of Rev1 catalytic activity, the

rominent CC to CG signature at position 530 was abolished, leavingnly the residual CC to CT transitions also observed at this site in WTcompare Fig. 5C to Fig. 4A and see Supplementary Table 4). Anotheronsequence was that C857T became the most prominent hotspot

12 (2013) 878– 889

in the rev1-AA spectrum (see Fig. 5C). Altogether, these data suggestthat the deoxycytidyltransferase activity of Rev1 is necessary forthe vast majority of (if not all) instances where C is inserted oppositeabasic sites, consistent with previous reports based on assays of TLSopposite a single, engineered abasic site in plasmid substrates [30]and analysis of unselected TLS events in a genetic reversion systemwithin excision repair deficient backgrounds [33].

3.6. In contrast to rev3 and rev1, pol32 cells retain a residualcapacity to insert C opposite abasic sites

Pol32 is an accessory subunit of both Pol � (a replicative poly-merase) and Pol � (a TLS polymerase) [42–44]. As mentionedpreviously, deletion of POL32 resulted in a TLS defect that was com-parable to that seen in rev3 and rev1 (see Fig. 2). But given thedual role of Pol32, we surmised that there might be differences inits mutation spectrum when compared to either rev3 or rev1. Wesequenced 48 independent pol32 isolates, each containing a singlemutation in CAN1 (see Fig. 6A). In contrast to the absence of CC to CGtransversions in rev3 and rev1, we observed 20 such substitutionsin pol32 (see Fig. 3A). 13 of these CC to CG transversions occurredat positions 530 or 718 (see Fig. 3C and Supplementary Table 4). Inaddition, the relative proportions of CC to CT, CC to CG, and all othermutations were significantly different compared to WT (P = 0.0004by Chi-square test, see Table 2). Thus, pol32 exhibited a mutationsignature that was unique among the various genetic backgroundsexamined.

The relative prominence of CC to CG transversions in pol32 ledus to test whether Rev1deoxycytidyltransferase activity was nec-essary for these substitutions. As already mentioned, loss of Rev1catalytic activity in the pol32 background resulted in a furthersignificant decrease of TLS activity compared to the pol32 singlemutant (see Fig. 2). We sequenced 23 independent isolates fromthe pol32 rev1-AA background, each bearing a single mutation inCAN1. As expected, CC to CG transversions were abolished (seeFigs. 3A and 6B). CC to CT transitions were by far the predomi-nant type of mutation, seen in 19 isolates. Eight of these eventswere observed at position 718 (see Fig. 3C), a site that also favoredinsertion of A opposite the abasic site in WT. Taken together, weconclude that the residual TLS in pol32 cells (evident when com-pared to rev3 or rev1) was attributable mostly to a capacity forinserting C opposite abasic sites, in a Rev1 catalysis-dependentmanner.

4. Discussion

4.1. An in vivo model system to investigate TLS bypass of lesionsin DNA

In this work, we describe the use of a subtelomeric ssDNAreporter system to define the roles of proteins involved in error-prone TLS bypass of abasic sites generated at random CC motifs inchromosomal DNA. Abasic sites are well-studied lesions in a vari-ety of systems. Using the ssDNA reporter, we have recapitulatedprevious findings on the roles of TLS proteins in yeast, thus validat-ing our overall approach. Additionally, we obtained evidence thatsequence context can strongly influence the relative likelihood ofA vs. C insertion opposite abasic sites, a possibility that had hardlybeen investigated previously. We also found that deletion of POL32results in a mutation signature entirely distinct from that of anyother genetic defect in TLS.

Yeast has a relatively small number of proteins that take partin TLS. Thus, it was straightforward to construct a set of relevantmutants to investigate the relative contribution of each proteintoward overall TLS activity to bypass abasic sites. In principle, the

K. Chan et al. / DNA Repair 12 (2013) 878– 889 885

Fig. 5. The mutation spectra of (A) rev3, (B) rev1, and (C) rev1-AA strains expressing APOBEC3G during temperature shift. All three genotypes were devoid of CC to CGtransversions. Deletion of either REV3 (A) or REV1 (B) resulted in a prominent CC to CT hotspot at position 718. In contrast, rev1-AA cells (C) exhibited frequent CC to CTt ion 53i sfera

sfirqmcTtowto

ransitions at position 857. In addition, CC to CT transitions were observed at positnsertion of C opposite abasic sites was entirely attributable to the deoxycytidyltran

ame approach can be applied to determine the TLS bypass speci-city of a large variety of lesions. In this study, we used only oneeporter gene (CAN1) to assess TLS bypass because the low fre-uency of TLS in the various mutant backgrounds required theutagenization of, and selection from, large numbers of TLS mutant

ells to obtain meaningful data. Under conditions of efficient WTLS, we had easily identified many instances of inactivating simul-aneous mutations in multiple reporter genes (i.e. mutation clusters

r showers) within the same mutant isolate [7]. In effect, TLS eventsere sampled at even larger numbers of different sequence con-

exts without having to select from an impractically large numberf mutagenized cells. Indeed, we have used this system to show that

0, whereas in WT cells this was a strong CC to CG hotspot. These data suggest thatse activity of Rev1.

TLS is necessary for bypass of sulfite-induced lesions at cytosinesin ssDNA [7], although the relative contributions of the various TLSproteins toward this type of lesion bypass have yet to be investi-gated.

The subtelomeric ssDNA reporter also has two key advantagesover conventional mutagenesis reporter systems. First, the single-strand state of the reporter DNA enables detection of mutagenesisarising from agents that are not reactive enough to damage dsDNA

appreciably, but might be able to form adducts in ssDNA. Suchagents usually exhibit only weak activity in conventional muta-genesis reporters [63]. Secondly, the known strandedness of thereporter DNA in this system enables unambiguous assignment

886 K. Chan et al. / DNA Repair 12 (2013) 878– 889

Fig. 6. The mutation spectra of (A) pol32 and (B) pol32 rev1-AA subtelomeric ssDNA reporter cells expressing APOBEC3G during temperature shift. (A) In contrast to all otherg and 7w ositio

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enotypes examined, pol32 cells exhibited CC to CG hotspots at both positions 530

ere abolished in pol32 rev1-AA cells. There were frequent CC to CT transitions at p

f the actual base change. In contrast, conventional mutagen-sis reporters typically are unable to distinguish whether angent induces a given mutation or its complement, and areeliant upon other experimental approaches to provide suchnformation.

.2. The importance of Rev1 catalytic activity

Our results provide clear evidence that Rev1 catalytic activitys required for the insertion of C opposite abasic sites randomlyenerated in chromosomal DNA, which is consistent with previ-us findings in yeast [30,33] and vertebrates [64,65]. When Rev1atalytic activity is removed, the overall frequency of TLS doesot decrease significantly compared to WT, i.e. the rev1-AA cellsre not defective for TLS opposite abasic sites, per se. However,he rev1-AA mutation spectrum is completely devoid of CC to CGransversions, indicating that the TLS bypass mechanism which iseft intact (requiring Rev1 structural function and Pol �) does notnsert C opposite abasic sites at a detectable frequency. Further, theoss of Rev1 catalytic activity does not have any apparent effect onhe ability to insert A opposite abasic sites, indicating that poly-

erase activities besides that of Rev1 are entirely responsible for insertion events.

As noted, abasic sites can be formed by enzymatic excision ofamaged bases [2]. One of the most commonly occurring damagedases is 8-oxo-7,8-dihydroguanine (8-oxoG), which results fromhe reaction of G with reactive oxygen species and can mispairith A [2]. 8-oxoG is excised by conserved DNA N-glycosylases,

.g. Ogg1 in yeast, to generate an abasic site [66]. Also, alkylatinggents are particularly reactive toward N7 of guanine, such thatdducts in this position destabilize the N-glycosidic bond, resultingn spontaneous depurination, i.e. abasic site formation [67]. Sincebasic sites resulting from damage to guanine are likely to be espe-ially common, it is plausible that evolution selected for an enzyme

pecifically with deoxycytidyltransferase activity (Rev1) to coun-eract the possible mutagenic effects. Such a hypothesis also woulde consistent with the notion that abasic sites are cognate lesionsor Rev1 [68].

18. Also, CC to CT transitions were relatively infrequent. (B) CC to CG transversionsn 718.

4.3. The nucleotide inserted opposite an abasic site is indicative ofthe set of TLS activities at work

By analyzing the outcomes of TLS events in various TLS defi-cient backgrounds at numerous randomly generated abasic sites,we have gained important insight into the underlying molecu-lar mechanisms. Sequence context can have a striking effect onthe choice of nucleotide inserted opposite an abasic site. Specifi-cally, abasic sites at position 530 of CAN1 tend to be bypassed byC insertion (resulting in CC to CG transversions), while abasic sitesat position 718 tend to be bypassed by A insertion (resulting inCC to CT transitions) (see Fig. 3A). The local sequence context ofposition 530 is 5′-AGTGATTGCCCAAGAAAACCA-3′ while the con-text of position 718 is 5′-ATTAGAAACCCGATAATGGCT-3′ (positions530 and 718 are underlined, respectively). As there are numerousdifferences between these two sequence contexts, further workis necessary to determine which sequence features influence thechoice of nucleotide insertion. Although Lawrence’s group hadobserved previously that varying the position of an engineeredabasic site by one base can result in statistically significant dif-ferences in the insertion frequencies of A vs. C [28,29], the effectsthat we have observed are much more pronounced. Differences insequence context also might be a factor underlying the Lawrencegroup’s observation of frequent (76%) insertion of C opposite theirmodel abasic sites [29] while Prakash’s group observed frequent(66%) insertion of A opposite their model abasic site [30].

Our results are summarized in Fig. 7. In WT (and rad30) cells(Fig. 7A), where TLS bypass of abasic sites is fully proficient, TLSinvolving Rev1 structural function and Pol � enables insertion ofA, while TLS involving Pol � and both Rev1 structural and cat-alytic functions enables insertion of C. When either REV3 or REV1is deleted (Fig. 7B), only a residual amount of low-frequency TLS(possibly by Pol � acting alone) that always inserts A opposite aba-sic sites is observed. Ablation of Rev1 catalytic function (Fig. 7C)leaves the Rev1 structural function- and Pol �-dependent pathway

intact, which explains the observation of only A insertions (and acomplete lack of C insertions) in rev1-AA cells. Deletion of POL32(Fig. 7D) leaves both Pols � and � as structurally incomplete com-plexes, resulting in a severe TLS defect. But in contrast to rev3 and

K. Chan et al. / DNA Repair 12 (2013) 878– 889 887

Fig. 7. Summary of the relative contributions of alternative TLS mechanisms toward mutagenesis at abasic sites. (A) In WT and rad30, TLS dependent on Pol � and thestructural function of Rev1 is necessary for the majority of A insertion events, while C insertion events require the presence of Pol � and fully functional Rev1. The relativefrequencies of the two outcomes are roughly equal. (B) In rev3 and rev1, C insertion events are not observed. The observed A insertion events are independent of Pol � andR presum� ts in ree mablyp plete

rtci

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ev1, and might represent low-frequency TLS bypass by a replicative polymerase ( and Rev1 structural function, are observed. The complete lack of C insertion evenvents in WT. (D) In pol32, both Pols � and � are structurally incomplete. This presuol32 rev1-AA, only low-frequency A insertion events are observed, involving incom

ev1, a readily detectable proportion of C insertions occur. Finally,he C insertions in pol32 cells are completely dependent on Rev1atalytic function (Fig. 7E), since only A insertion events are foundn the pol32 rev1-AA double mutant.

Taken together, we conclude that in cells fully proficient for TLS,ertain sequence contexts (e.g. around C530) tend either to promote

he catalytic activity of Rev1 to insert C, or inhibit the activity of Pol

to insert A, or some combination of both effects. Conversely, otherequence contexts, such as near C718, either promote the activity ofol �, inhibit the catalytic activity of Rev1, or some combination of

ably Pol �) acting alone. (C) In rev1-AA, only A insertion events, which require Polv1-AA implies that Rev1’s deoxycytidyltransferase function is responsible for such

results in low-frequency, but observable TLS events that insert either A or C. (E) In Pol � and Rev1 structural function.

both. We speculate that contexts favoring C insertion tend to stabi-lize Rev1 (by some combination of hydrogen bonding, salt bridges,and van der Waals interactions) in a configuration favorable forcatalysis, while the opposite is true for contexts favoring A inser-tion. As with any mutagenesis reporter system, selection influenceswhich mutations are recovered most frequently and necessarily

leads to the assessment of only a subset of all possible mutationswithin a reporter gene (reviewed in [69]). Thus, it is important toextend this work to a genomewide scale, sampling TLS events overa larger number of sequence contexts that are not under selection

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ressure as CAN1 inactivation was. This could identify the sequenceeterminants that influence which contexts favor one type of TLSvent over the other.

.4. Abasic site bypass within long regions of ssDNA in cancerenomes

Recent work has shown that mutations at cytosines in 5′-TC-′ motifs were significantly enriched in sequenced tumors fromultiple types of cancer [9–11]. Further, clusters of multiple pointutations (C to G/T substitutions) that all occurred at TC motifsere identified [10,11]. These observations suggest that at someoint during tumorigenesis, long stretches of ssDNA were exposedo deamination by at least one enzyme among the TC-specific sub-lass of APOBEC deaminases (reviewed in [70]). Presumably, UNGlycosylases then excised the resulting uracils to generate abasicites, which were bypassed in an error-prone manner by TLS.

Humans possess a larger complement of TLS polymerases thaneast [12–14]. It might be possible to carry out a detailed analysis ofhe role of each human TLS protein in the bypass of random abasicites in vivo by heterologous expression of each human TLS poly-erase (or combinations of multiple human TLS polymerases), in a

east reporter deleted for the yeast TLS polymerases while express-ng APOBEC3G. If this heterologous expression were successful,

utation spectrum analysis to infer the roles of individual humanLS proteins should be possible. Such work could shed importantew light on molecular mechanisms of carcinogenesis.

. Conclusions

Genomic DNA is susceptible to mutation arising from dam-ge caused by endogenous and environmental agents [1]. Sinceenomewide sequencing of human cancers already has revealedhe pervasive, distinctive mutagenic signature of endogenoussDNA damaging agents (the APOBEC cytosine deaminases) [9–11],e propose that other agents also would have the opportunity to

eact with long stretches of ssDNA at some point(s) during carcino-enesis. Having validated our experimental approach by analyzingbasic sites, the ssDNA reporter strains now can be used to iden-ify and characterize ssDNA-specific mutagens, i.e. agents that areot reactive enough to damage dsDNA significantly, but poten-ially can react with ssDNA in vivo to form lesions that lead to

utation fixation. Detailed understanding of the underlying muta-enic mechanisms could be obtained by determining the TLS bypasspecificity of such lesions. In turn, this knowledge would help deci-her the complex superimposition of myriad DNA damage, repair,nd damage tolerance processes that acted to create any given can-er genome [71].

onflict of interest statement

The authors declare that there are no conflicts of interest.

cknowledgments

We thank Drs. T.A. Kunkel, D.R. Menendez, T.T. Nguyen, and S.A.oberts for critical reading of the manuscript. This work was sup-orted by the Intramural Research Program of the National Institutef Environmental Health Sciences (NIH, DHHS) project ES065073 toAR.

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