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d n a r e p a i r 7 ( 2 0 0 8 ) 597–604

avai lab le at www.sc iencedi rec t .com

journa l homepage: www.e lsev ier .com/ locate /dnarepai r

Rev7, putative subunit of hPol�, plays a critical role inurvival, induction of mutations, and progression through-phase, of UV(254 nm)-irradiated human fibroblasts�

ristin McNally1, Jessica A. Neal, Terrence P. McManus,. Justin McCormick, Veronica M. Maher ∗

arcinogenesis Laboratory, Cell and Molecular Biology Program, Department of Microbiology & Molecular Genetics, and Department ofiochemistry &Molecular Biology, Michigan State University, East Lansing, MI 48824-1302, USA

r t i c l e i n f o

rticle history:

eceived 24 October 2007

eceived in revised form

0 December 2007

ccepted 21 December 2007

ublished on line 4 March 2008

eywords:

ev7

olymerase zeta

ranslesion synthesis

ltraviolet radiation

AD2B

ev3

a b s t r a c t

Translesion synthesis (TLS) refers to mechanisms by which specialized DNA polymerases

incorporate nucleotides opposite fork-blocking lesions and extend replication until stan-

dard replicative polymerases take over. The first eukaryotic TLS polymerase discovered, S.

cerevisiae Pol�, consists of catalytic subunit Rev3 and non-catalytic subunit Rev7. Human

homologs of these two proteins have been identified. Studies by Lawrence, Maher, and col-

leagues comparing UV(254 nm)-irradiated human fibroblast cell strains expressing high levels

of hRev3 antisense to their normal parental strains demonstrated that there was no dif-

ference in cell survival, but that the frequency of UV-induced mutations in the derivative

strains was 10-fold lower than that of the parental strains, indicating that hRev3 plays a

critical role in such mutagenesis. To examine the role of hRev7 in TLS, we generated human

fibroblasts expressing hRev7 siRNA, identified two derivative cell strains with significantly

reduced levels of hRev7, and compared them to their parental strain and a vector control for

cell survival, induction of mutations, and ability to traverse the cell cycle following expo-

sure to UV radiation. Cells with reduced hRev7 were ∼2-times more sensitive to UV-induced

cytotoxicity than the controls, indicating that unlike hRev3, hRev7 plays a protective role

for cells exposed to UV radiation. When these cell strains were assayed for the frequency of

mutations induced by UV in their HPRT gene, cell stains with reduced hRev7 were 5-times

less sensitive to UV-induced mutagenesis than control strains. In addition, when these four

strains were synchronized at the G1/S border, released from the block, UV-irradiated, and

allowed to traverse the cell cycle, the rate of progression through S-phase of the cell strains

with reduced hRev7 was significantly slower than that of the control strains. These data

strongly support the hypothesis that hRev7 is required for TLS past UV-photoproducts, and

together with hRev3, comprise hPol�.

� Disclosure: Materials used were not from other published papers. Th∗ Corresponding author at: Carcinogenesis Laboratory, Food Safety anI 48824-1302, USA. Tel.: +1 517 353 7785; fax: +1 517 353 9004.

E-mail address: maher@msu.edu (V.M. Maher).1 Present address: Laboratory of Persistent Viral Diseases, NIAID/NIH568-7864/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.dnarep.2007.12.013

© 2008 Elsevier B.V. All rights reserved.

e data in this manuscript have not been published elsewhere.d Toxicology Building, Michigan State University, East Lansing,

, Rocky Mountain Laboratories, Hamilton, MT 59840, USA.

7 ( 2

598 d n a r e p a i r

1. Introduction

Human cells are continually exposed to endogenous andexogenous DNA damaging agents, many of which create fork-blocking lesions. If DNA replication past such lesions cannottake place, this can lead to cell death, nevertheless replicationpast such lesions can result in mutations. Because mutationsplay a crucial causal role in the development of cancer, it isimportant to examine processes that produce them.

Human cells have efficient, error-free repair pathways forexcising DNA fork-blocking lesions from either strand of theirDNA. They also possess cell cycle checkpoints [1], some ofwhich, when activated, provide additional time for excisionrepair to occur before the replicative polymerases encounterfork-blocking lesions, such as UV-induced pyrimidine dimers.In spite of these protective processes, replication forks stillencounter lesions. Cells have evolved damage tolerance mech-anisms to cope with such lesions, viz., translesion synthesisand damage avoidance pathways. Such methods of dealingwith fork-blocking damage have been, and continue to beactively examined. Overviews summarizing in detail suchareas of research can be found in reference [2].

Translesion synthesis in both prokaryotes and eukaryotesinvolves specialized DNA polymerases capable of incorpo-rating nucleotides directly across from fork-blocking DNAlesions. This insertion step can be error-free or error-prone,depending upon (1) the type of DNA lesion encountered, (2)the specialized polymerases involved, and (3) the sequencecontext surrounding the site of the damage. Insertion of anucleotide or nucleotides by one or other such polymerases isfollowed by extension, i.e., the addition of nucleotides beyondthe site of the blocking lesion. This latter step also involvesTLS DNA polymerases. Such extension beyond the damageis necessary if the high fidelity replicative DNA polymerasesare to resume their function. Thus, TLS is a two-step processwhereby specialized DNA polymerases, with relaxed fidelity,incorporate and/or extend nucleotides at sites of fork-blockingDNA damage, allowing DNA replication to continue, but oftenintroducing mutations.

Reports and summaries of the discovery of many transle-sion synthesis polymerases, first in S. cerevisiae, and later inmammalian cells, can be found in the cited references (seefor example, [3–6]). However, many aspects still remain to beclarified. Pol� was found using S. cerevisiae cells whose spe-cific mutated phenotypes could not be reverted to wild type byexposure to mutagenic agents. Genes that complemented thedeficiencies in such strains of S. cerevisiae, i.e., allowed themto revert, were identified and subsequently shown to code forproteins that allow replication past fork-blocking DNA dam-age [7,8]. For example, the yeast Rev3 protein was found toexhibit polymerase activity in primer extension assays in vitro.The addition of yeast Rev7 to such assays enhanced the poly-merase activity of Rev3 over 20-fold. Together, Rev3 and Rev7were recognized as constituting yeast Pol� [9].

Genes coding for the human homologs of yeast Rev3 [10–13]

and Rev7 [14] were subsequently identified. By using anti-sense directed against hRev3 mRNA, Lawrence, Maher, andtheir colleagues [10,15] demonstrated that hRev3, the putativecatalytic subunit of hPol�, is critically involved in generating

0 0 8 ) 597–604

UV-induced mutations in diploid human fibroblasts. Theseresults indicate that hRev3 is essential for a mutagenic pro-cess involving DNA lesions that interfere with replication, justas yeast Rev3 is. The hRev3 protein of human cells, a predicted353 kDa molecule [10], has not yet been isolated, but the non-catalytic subunit, hRev7, a much smaller molecule, has beenisolated [14].

The present study was carried out to test the hypothe-sis that hRev7, the putative non-catalytic subunit of hPol�, isalso involved in human cell mutagenesis. For such a study,an approach similar to that used for investigating the role ofhRev3 was employed, but instead of using antisense RNA toblock expression of the target protein, siRNA against hRev7was used to reduce the level of this protein in human fibrob-lasts. The fact that antibodies capable of detecting very lowlevels of hRev7 protein were available allowed us to identifyindependent cell strains in which the level of hRev7 proteinhad been greatly reduced by siRNA. Comparing the resultsobtained using these cell strains with those obtained usingtheir parental human fibroblasts and a vector control strainallowed us to demonstrate that hRev7, the non-catalytic sub-unit of hPol�, plays a role in the survival of UV-irradiatedhuman cells, and has a significant role in UV-induced mutage-nesis. Using these human cell strains, we also demonstratedthat reduction in the expression of hRev7 impedes the cells’ability to progress through S-phase.

2. Materials and methods

2.1. Cell culture

Cells were grown in Eagle’s minimum essential medium,supplemented with 0.2 mM l-aspartic acid, 0.2 mM l-serine,1 mM sodium pyruvate, 10% supplemented calf serum(HyClone), 100 units/ml penicillin, 100 �g/ml streptomycin,1 �g/ml hydrocortisone and 1 �g/ml tetracycline.

2.2. Cell strains

The parental human cell strain used for these studies, des-ignated MSU-1.2.9N.58, was derived from the cell strainMSU-1.2, a spontaneous derivative of the infinite life span cellstrain MSU-1.1, whose origin from the foreskin-derived froma normal neonate and subsequent acquisition of an unlim-ited life span in culture has been described [16]. MSU-1.2 cellsare near-diploid, chromosomally-stable, and grow vigorouslyas a result of expressing their endogenous gene for platelet-derived growth factor.

2.3. Derivation of cell strains with reduced hRev7

Oligonucleotides designed to target hRev7 mRNA wereannealed to a complementary oligonucleotide according to themanufacturer’s protocol (Ambion). Using T4 DNA ligase (NewEngland Biolabs), annealed-oligonucleotides were ligated into

the pSilencer3.1 vector (Ambion), which includes the genecoding for puromycin resistance, and purified. The parentalMSU-1.2.9N.58 cells were transfected with such siRNA vectors,using Lipofectamine (Invitrogen) according to the manufac-

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urer’s protocol, and stable transfectants were selected andaintained in medium containing 1 �g/ml puromycin.

.4. Preparation of nuclear protein extracts andestern blot analysis

ubconfluent monolayers of cells were washed with ice-coldhosphate buffered saline (PBS), scraped from the 150-mm-iameter dishes in 1 ml of lysis buffer A (10 mM HEPES, 10 mMCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF), and

ncubated on ice for 15 min. 10% NP-40 (62 �l) was added toach sample of lysed cells, and the samples were vortexed for0 s, and centrifuged for 30 s at 10,000 rpm, 4 ◦C. The super-atant containing cytoplasmic proteins was removed, and theuclear pellet was washed once in 1 ml of buffer A contain-

ng 10% NP-40. Nuclear proteins were extracted by disruptionf the nuclei in 40 �l of lysis buffer C (20 mM HEPES, 0.4 MaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM sodium ortho-anadate, 1 mM PMSF) and incubated on ice for 15 min withortexing every 5 min. Nuclear extracts were centrifuged formin at 16,000 rpm, 4 ◦C. The supernatants, which contained

he nuclear proteins, were saved. Protein was quantified usinghe Bradford method (Pierce). Protein lysates were subjectedo gel electrophoresis using 14% SDS-polyacrylamide, trans-erred to a PDVF Immobilon membrane (Millipore), and probedith a 1:600 dilution of a custom-made (Bethyl) rabbit poly-

lonal antibody raised against the C-terminal 19 amino acidsf the human Rev7 protein. The membrane was probed with a:7500 dilution of goat anti-rabbit secondary antibody (Sigma)nd visualized using SuperSignal chemiluminescent detec-ion reagent (Pierce). Equal protein loading was confirmed byrobing with a 1:10,000 dilution of a rabbit Ku80 antibody

Santa Cruz) and a 1:10,000 dilution of anti-rabbit secondaryntibody (Santa Cruz).

.5. Assay of UV cytotoxicity

he cytotoxic effect of UV(254 nm) radiation was determined byssaying the colony-forming ability of the cells as described17]. Briefly, cells in exponential growth were detachedrom the dishes with trypsin, plated at cloning densities100–600 cells per 100-mm-diameter dish), and allowed 12 h forttachment. The cells were rinsed twice with PBS, the excessBS was removed, and the cells were irradiated with the desig-ated doses of UV(254 nm) as described [17]. Immediately after

rradiation, the cells were given fresh culture medium. Theulture medium was renewed 24 h after irradiation and againfter 7 days. After 14 days, the resulting clones were stainedith crystal violet. Cell survival was determined by compar-

ng the cloning efficiency of the irradiated cells with that ofhe sham-irradiated control cells. Cell survival at each doseas expressed as a percent of the cloning efficiency of thenirradiated control cells for each cell strain.

.6. Assay for frequency of UV-induced mutations

he mutagenic effect of UV radiation in each strain wasetermined from the frequency of cells that lost expression ofhe HPRT gene and, therefore, were resistant to 6-thioguanine.s described [17], sufficient sets of cells plated at densities

8 ) 597–604 599

of 0.5–1.5 × 106 cells per 150-mm-diameter dish, were used,to ensure at least 1 × 106 surviving target cells per dose. Cellswere allowed 12 h for attachment, then rinsed twice withPBS, UV-irradiated at the designated doses, and immediatelycovered with fresh culture medium. The culture medium wasrenewed 24 h after irradiation, and the cells were allowedto replicate for 4 days. Cells were then detached usingtrypsin, pooled, plated at densities of 0.5–1.0 × 106 cells per150-mm-diameter dish, and allowed to grow exponentiallyfor 4 additional days in order to deplete the pre-existingwild-type HPRT protein. Cells were then detached usingtrypsin, and plated at a density of 500 cells/cm2 in mediumcontaining 40 �M 6-thioguanine (TG) to select for cells lackingfunctional HPRT protein. At the same time, a portion of cellsfrom each population was plated in non-selective medium ata density of 100 cells per 100-mm-diameter dish to assay thecolony-forming ability of the cells at the time of selection.The medium on these cells was renewed after 7 days. After14 days, the colonies that had formed were stained withcrystal violet, and the frequency of 6-TG-resistant colonieswas calculated using the cloning efficiency of the cells at thetime of selection. The induced frequencies for each cell strainwere calculated by subtracting the background frequencies inthe sham-irradiated control populations that accompaniedeach experiment.

2.7. Hypoxanthine phosphoribosyltransferase (HPRT)mutation spectrum analysis

HPRT-defective colonies were obtained essentially asdescribed above for the UV-induced mutation frequencyprotocol, except that populations were kept independentto avoid sibling mutations. The TG-resistant mutant cloneswere isolated by treating with trypsin, and each independentmutant was subjected to reverse transcription, followed bytwo rounds of PCR to amplify the HPRT coding region. PCRproducts were purified (Qiagen) and sequenced at the MSUmacromolecular structure facility to determine the specificmutation in the coding region of HPRT. Only base substitu-tions at adjacent pyrimidines that resulted in an amino acidchange were considered UV-induced mutations.

2.8. Cell synchronization and flow cytometry analysis

Each cell strain was plated at a density of 0.2 × 106 cells per100-mm-diameter dish and allowed 12 h for attachment. Cellswere then re-fed with complete culture medium containinglovastatin at a final concentration of 60 �M to synchronize thecells in early G1 phase. After 12 additional hours of incuba-tion, the medium containing lovastatin was removed, the cellswere washed twice with PBS, and culture medium containingaphidicolin at a final concentration of 2 �g/ml and mevalonicacid at a final concentration of 6 mM was added to the dishesfor 12 h to synchronize the cells at the G1/S border. The cellswere released from the aphidicolin/mevalonic acid block byrinsing twice with PBS, and immediately irradiated with the

designated doses of UV as described [17]. At the designatedtimes post-irradiation, cells were detached using trypsin, fixedin 80% ethanol, and stained with a propidium iodide solu-tion (PBS, 1 mg/ml propidium iodide, 10% Triton X-100, 0.5 mM

7 ( 2 0 0 8 ) 597–604

Fig. 1 – Western blot analysis of hRev7 protein levels in thecell strains. A polyclonal antibody against hRev7 was usedto analyze the level of hRev7 protein in nuclear lysatesextracted from the parental cells (P), the vector control cells(VC), and two derivative clones expressing a transfectedsiRNA targeted against hRev7, viz., clones 2–2 and 2–6. The

600 d n a r e p a i r

EDTA, 10 mg/ml RNase A) for cell cycle analysis by flow cytom-etry. Asynchronously-growing cells were assayed in parallelexperiments.

2.9. Statistical methods

To compare the slopes of the curves in Fig. 2A and B, a regres-sion model was used. The slopes indicate the effect of UV onthe survival (A) of the cells’ colony-forming ability and on theirfrequency of UV-induced mutants (B). The data were takenfrom a series of experiments that were treated as blocks inthe regression analysis.

For comparing the types of mutations induced (see Table 1),the sparse categories, i.e., C → G and T → G, were collapsedfor the chi-square analysis. They showed no statisticallysignificant difference between the clones and the compar-ison group (P-value = 0.60). Other categories were collapsedto reduce the number of table cells analyzed with smallexpected frequencies.

3. Results

3.1. Efficient reduction of hRev7 protein using siRNA

The parental cell strain, designated MSU-1.2.9N.58, was trans-fected with a vector expressing an siRNA targeted againsthRev7 and also carrying the gene for puromycin resistanceto allow selection of transfectants. As a control, we similarlytransfected the parental cells with a vector containing thisselectable marker and an siRNA that has no significant homol-ogy to any human gene sequence. Puromycin-resistant cloneswere isolated and expanded. Nuclear protein extracts from theparental cells, vector control transfectants, and from candi-date transfectants that received the siRNA against hRev7 wereprepared and analyzed by Western blotting for their level ofexpression of hRev7. Fig. 1 shows a representative Westernblot. Lane 1 shows hRev7 protein from the parental cell strain(P), migrating as expected for a 24 kDa protein. Lane 2 showshRev7 protein from the vector control cell strain (VC). Lanes 3and 4 were loaded with protein from two clones, designated2–2 and 2–6, that had been transfected with a vector express-ing hRev7 siRNA. No hRev7 protein could be observed in thelatter two clones (Fig. 1). However, when the Western blotswere allowed ≥24 h of exposure, a very low level of hRev7protein could be detected on the blots (data not shown). Themorphology of clones 2–2 and 2–6 did not differ from that ofthe parental or the vector control cells. Neither did the rate ofgrowth in culture of these two cell strains differ from that oftheir parent or the vector control cells (data not shown).

3.2. Effect of reduction of the level of hRev7 protein onthe sensitivity of the cell strains to the cytotoxic effect ofUV(254 nm) radiation

To examine the sensitivity of these cell strains to the cytotoxic

effect of UV, we irradiated the parental cells, the vector controlcells, and the two cell clones virtually devoid of hRev7 proteinand assayed them for survival of colony-forming ability. Asshown in Fig. 2A, the parental cell strain and the vector control

arrowhead on the right indicates the location of a 22 kDamarker. Ku80 was used as the loading control.

strain exhibited identical sensitivity to the cytotoxic effect ofUV(254 nm) radiation. The dose required to reduce their survivalto 37% that of unirradiated cells was 12 J/m2. The survival ofclones 2–2 and 2–6 were identical to each other, but both were1.7 times more sensitive to UV-induced cell killing than thecontrol cells. The dose required to reduce their survival to 37%that of unirradiated cells was only 8.5 J/m2. These differenceswere shown to be statistically significant. The data indicatethat hRev7 plays a protective role in survival of cells exposedto UV irradiation.

3.3. Effect of reduced hRev7 protein on the frequencyof UV-induced mutations

The frequency of mutations induced by UV in the HPRT geneof these four cell strains was determined using resistance to6-thioguanine as an indicator of cells with a mutation in theirHPRT gene. As shown in Fig. 2B, the frequency of mutationsinduced by UV in the parental cells and vector control cellsexpressing hRev7 were identical. The dose that reduced theirsurvival to 37%, i.e., 12 J/m2, induced an HPRT mutation fre-quency of ∼135 × 10−6. In contrast, the two clones with greatlyreduced expression of hRev7 protein showed a statisticallysignificant decrease in the frequency of UV(254 nm)-inducedmutations (P-value < 0.001 for comparison of slopes). The fre-quency of mutants induced by 12 J/m2 in clones 2–2 and 2–6was only 32 × 10−6 clonable cells. At a dose that reduced theirsurvival to 37%, i.e., 8.5 J/m2, the frequency of induced mutantswas ∼20 × 10−6, significantly lower than the 135 × 10−6 seenin the two control cell strains. This significant reduction inthe frequency of UV-induced mutations in cells with reducedhRev7 indicates that hRev7 plays an important role in UV-induced mutagenesis in human fibroblasts.

3.4. Effect of decreased hRev7 protein expression onkinds of UV-induced mutations

The kinds of base substitutions induced by UV in the codingregion of the HPRT gene of cells expressing or not expressing

hRev7, as determined by nucleotide sequencing, are shown inTable 1. The data report kinds of mutations induced in the twoclones extremely deficient in expression of hRev7 (clones 2–2and 2–6) and those induced in the comparison group (parent

d n a r e p a i r 7 ( 2 0 0 8 ) 597–604 601

Fig. 2 – Effect of reduced expression of hRev7 on thesurvival of cell strains exposed to UV(245nm) radiation andon the frequency of UV-induced mutations. (A) Clones 2–2and 2–6 (closed symbols), which have greatly reduced levelsof expression of hRev7 protein, along with their parentalstrain and a vector control strain (open symbols) wereUV-irradiated and assayed for cell survival as determinedby colony-forming ability. Some data points have beenoffset slightly to make them visible. The lines representleast squares lines. (B) The frequency of UV-inducedmutations in the HPRT gene of these four cell strains wasdetermined by resistance to 6-thioguanine. The frequencyof 6-thioguanine-resistant cells was calculated using thecloning efficiency of cells at the time of selection, whichaveraged ∼44% for the parent and the vector control cells,and ∼25% for clones 2–2 and 2–6. Induced frequencies werecalculated by subtracting the background frequenciesobserved in sham-irradiated populations. For the vectorand parental control cells, these values werealways < 11 × 10−6. For clone 2–2, they ranged from0–18 × 10−6, with the majority being 0–4 × 10−6. For clone2–6, they ranged from 8–25 × 10−6, with the majority being8–11 × 10−6. Some data points have been offset slightly tomake them visible. The lines represent least squares lines.

Table 1 – Kinds of UV-induced base substitutions in theHPRT gene of cells with normal or greatly decreasedlevels of hRev7

Base changes Parent and vectorcontrol

Clone 2–2 andclone 2–6

C → T 25 (47.2%) 19 (45.2%)T → C 6 (11.3%) 2 (4.8%)T → A 8 (15.1%) 11 (26.2%)C → A 10 (18.9%) 7 (16.7%)C → G 4 (7.5%) 1 (2.4%)

T → G 0 2 (4.8%)

Total 53 (100%) 42 (100%)

and vector control). [A table detailing the kinds and showingthe context of 95 independent UV(254 nm)-induced mutations inHPRT of these two sets of human skin fibroblasts can be foundin the Supplement]. There was no statistically significant dif-ference in the kinds of mutations induced in the parentaland vector control cells compared to those induced in thetwo derivative cell strains, virtually devoid of hRev7 protein.These data indicate that reducing the level of hRev7 results ina decrease in the frequency of UV-induced mutations withoutaltering the types of base substitutions generated.

3.5. Evidence that reduction of expression of hRev7protein in human fibroblasts results in a UV-induced delayin traversing S-phase

To assess potential effects of decreased hRev7 on cell cycleprogression after UV irradiation, the parental and vectorcontrol cell strains and the two clones with greatly reducedhRev7 were synchronized at the G1/S border as described,released from the block, and immediately UV-irradiated with12 J/m2, the dose determined to reduce the survival of normalcells to ∼37% and that of the hRev7-deficient cells to ∼20%(see Fig. 2A). Independent synchronized populations of theseUV-irradiated cells were assayed by flow cytometry immedi-ately following release from the G1/S block (0 h), or after 10 hor 16 h. As shown in Fig. 3A, at the time of release from thereplication block (0 h), all four strains were synchronized atthe G1/S border. Ten hours later, the parental (P) and vectorcontrol cells (VC) were predominantly in S-phase and G2phase. In contrast, after 10 h, cell strains 2–2 and 2–6 werestill predominantly in G1 and S-phase, i.e., their cell cycleprogression was greatly delayed, compared to that of the twocontrol strains. The data from cells assayed 16 h followingUV irradiation show that the cell strains with reduced hRev7were still delayed in S-phase, compared to their parentalstrain and the vector control cell strain.

These four cell strains were similarly irradiated with12 J/m2 while growing asynchronously and assayed byflow cytometry. As shown in Fig. 3B, UV-irradiation ofasynchronously-growing cells with decreased expression ofhRev7 also delayed their progression through S-phase com-pared to control cells. As a control, we synchronized the

parental strain and the derivative strains with reduced hRev7,released them from the G1/S block, as described above, butdid not expose them to UV radiation. The subsequent flowcytometric analysis (Fig. 3C) demonstrated that all three unir-

602 d n a r e p a i r 7 ( 2 0 0 8 ) 597–604

Fig. 3 – Flow cytometry analysis of UV-irradiated cells. (A) Cell strains with reduced levels of hRev7 [2–2 and 2–6], theirparental strain [P], and a vector control transfectant [VC] were synchronized at the G1/S border as described, andUV-irradiated immediately after release from synchrony. The cell strains were assayed by flow cytometry for DNA content at0 h, 10 h, and 16 h post-irradiation. (B) These four cell strains were UV-irradiated while growing asynchronously and wereassayed by flow cytometry for DNA content at 0 h and 10 h post-irradiation. (C) As a control, clones 2–2, 2–6, and theirparental strain were synchronized at the G1/S border, as above, released from synchrony, but not exposed to UV, and

rom

analyzed by flow cytometry at 0 h, 4 h, and 6 h after release f

radiated cell strains proceeded through the cell cycle at anequal rate. Taken together, the data in Fig. 3 indicate that cellswith reduced hRev7 replicate their DNA more slowly in thepresence of UV-induced DNA damage.

4. Discussion

A role for hRev7 as the non-catalytic subunit of Pol� was sug-gested by its homology to the yeast Rev7 protein, as well as itsphysical interaction with human Rev3 in a yeast-two-hybridassay [14]. The results of our study establish a functional rolefor hRev7 in UV-induced mutagenesis, and what is more, a rolein the survival of colony-forming ability. The fact that at everydose of UV radiation, the cells with greatly decreased levels ofhRev7 protein exhibited ∼5-fold lower frequency of inducedmutations than the control cell strains is strong evidencethat in human cells the hRev7 protein plays a role in error-prone TLS past UV-induced DNA photoproducts, as does yeastRev7.

The mutagenesis data from the present study, using humanfibroblasts that have greatly reduced levels of hRev7 protein,confirm and greatly strengthen the results obtained previ-ously in this laboratory using similar cell lines and antisenseagainst hRev3 [10,15]. The ∼5-fold decrease in UV-inducedmutation frequency observed in cells virtually devoid of hRev7

is similar to the decrease in frequency observed previously inthis laboratory using human fibroblasts expressing antisensehRev3 [10,15]. These data strongly support the hypothesis thathRev3 and hRev7 function in the same pathway, most likely as

the G1/S block.

catalytic and non-catalytic subunits, respectively, of humanpolymerase �.

Our results demonstrating that human fibroblasts withreduced hRev7 are more sensitive to the cytotoxic effects ofUV radiation than the control strains (Fig. 2A) support theresults obtained by Cheung et al. [18], which demonstratedthat nasopharyngeal carcinoma cell lines with reduced hRev7are also more sensitive to the cytotoxic effects of specific DNAdamaging anticancer drugs than the control cells. The factthat reduction of hRev7 results in increased sensitivity to thecytotoxic effects of various DNA damaging agents in both nor-mal and cancer cells, underscores the importance of hRev7 forprotecting human cells from DNA damage.

In addition to sharing a high degree of amino acid sequencesimilarity to the yeast Rev7 protein, hRev7 also has a highdegree of similarity to the mitotic checkpoint protein hMAD2,and thus is also referred to as hMAD2B [14,19]. In fact, hRev7has been shown to interact with hMAD2 and furthermore, toinhibit the anaphase-promoting complex by binding to acti-vators Cdh1 and Cdc20 in Xenopus extracts, suggesting a rolefor hRev7 in regulating the mitotic checkpoint [14,20,21]. Onemight hypothesize that the sensitivity to the cytotoxic effectsof UV we observe in our human fibroblasts reflects interfer-ence with the mitotic checkpoint response of UV-irradiatedcells. However, Cheung et al. [18] found that reducing hRev7 innasopharyngeal carcinoma cells had no effect on their mitotic

checkpoint response. Therefore, we consider it unlikely thatthe increased sensitivity to the cytotoxic effects of UV radi-ation that we observe in human fibroblasts with reducedhRev7 results from an aberrant mitotic checkpoint response.

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evertheless, further experimentation could be conductedo specifically determine whether or not hRev7 affects the

itotic checkpoint of human fibroblast cells in the presencef DNA damage.

In addition to decreasing the survival of UV-irradiateduman fibroblast strains, reduced levels of hRev7 also resulted

n a marked decrease in their rate of progression through theNA synthesis phase of the cell cycle (Fig. 3). This suggests

hat for human cells with reduced expression of hRev7, andherefore with reduced hPol�, the presence of DNA damageuring S-phase presents a major problem for DNA replica-ion. If cells with decreased expression of hRev7 were simplynable to resume DNA replication as a result of impaired TLS,his situation would be expected to result in replication forkreakdown and ultimately lead to cell death. It is always pos-ible that replication arrest due to impaired TLS accounts forhe increase in sensitivity to the cytotoxic effects of UV thate observe when cell strains with reduced hRev7 are irradi-ted. However, previous results in our laboratory using humanells expressing antisense against hRev3, the catalytic subunitf hPol�, demonstrated that such cells did not differ from thearental cells in sensitivity to the cytotoxic effects of UV [15].aken together, these results suggest that in our clones witheduced hRev7, factors other than impaired TLS contribute tohe observed increase in sensitivity to the cytotoxic effects ofV.

In 2005, a study by Bi et al. [22], demonstrated mousembryonic fibroblasts lacking Pol� were unable to recover frombenzo[a]pyrene diol epoxide (BPDE)-induced S-phase check-oint and, in addition, were more sensitive to the cytotoxicffects of BPDE than were the wild-type cells. The fact thate found a clear indication of a UV-induced delay in S-phase,nd an increase in sensitivity to the cytotoxic effects of UVn cell strains with reduced hRev7, suggests that there is aimilar requirement for hRev7 in recovery from a UV-induced-phase checkpoint as there is for Pol� in a BPDE-induced S-hase checkpoint.

In summary, our mutagenesis data demonstrate thatRev7, like hRev3, is required for TLS past UV photoproductsnd is causally involved in producing the mutations that resultrom such TLS. These data strongly support the hypothesishat hRev7, together with hRev3, comprise hPol�. In addition,ur data demonstrate a requirement for hRev7 in protectinguman fibroblasts from the cytotoxic effects of UV-inducedNA damage that was not found in human fibroblast cellsxpressing antisense against hRev3. This suggests that therotective role of hRev7 for cells exposed to UV radiation is

ndependent of the requirement for hRev7 in hPol�-dependentLS.

onflict of interest

one declared.

cknowledgements

e thank Dr. Christopher Lawrence of the Department of Bio-hemistry and Biophysics at the University of Rochester for hiselpful advice, insight, and constant interest in our research.

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We also thank Dr. Katheryn Meek, Department of Pathobiology& Diagnostic Investigation at Michigan State University, forher advice and encouragement during this research, and Dr.Dennis Gilliland, Co-director of the Center for Statistical Train-ing and Consulting at Michigan State University for statisticalanalysis of the data. A special note of thanks to Andrew McCoyand James Reinhart for their assistance with experiments.This research was supported by Grant CA91490 awarded toV.M.M. from the National Cancer Institute of NIH.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.dnarep.2007.12.013.

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