Proliferating cell nuclear antigen is protected from degradation by forming a complex with MutT Homolog2.

Abstract

Proliferating cell nuclear antigen (PCNA) has been demonstrated to interact with multiple proteins involved in several metabolic pathways such as DNA replication and repair. However, there have been fewer reports about whether these PCNA-binding proteins influence stability of PCNA. Here, we observed a physical interaction between PCNA and MutT homolog2 (MTH2), a new member of the MutT-related proteins that hydrolyzes 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP). In several unstressed human cancer cell lines and in normal human fibroblast cells, PCNA and MTH2 formed a complex and their mutual binding fragments were confirmed. It was intriguing that PCNA and MTH2 were dissociated dependent on acetylation of PCNA, which in turn induced degradation of PCNA in response to UV irradiation, but not in response to other forms of DNA-damaging stress. To further explore the link between dissociation of PCNA-MTH2 and degradation of PCNA, RNAi against MTH2 was performed to mimic the dissociated status of PCNA to evaluate changes in the half-life of PCNA. Knockdown of MTH2 significantly promoted degradation of PCNA, suggesting that the physiological interaction of PCNA-MTH2 may confer protection from degradation for PCNA, whereas UV irradiation accelerates PCNA degradation by inducing dissociation of PCNA-MTH2. Moreover, secondary to degradation of PCNA, UV-induced inhibition of DNA synthesis or cell cycle progression was enhanced. Collectively, our data demonstrate for the first time that PCNA is protected by this newly identified partner molecule MTH2, which is related to DNA synthesis and cell cycle progression.

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Proliferating Cell Nuclear Antigen Is Protected from
Degradation by Forming a Complex with MutT Homolog2*□
Receivedforpublication,March10,2009,andinrevisedform,April30,2009 Published,JBCPapersinPress,May6,2009,DOI10.1074/jbc.M109.015289
Yu Yu‡1, Jian-Ping Cai§1, Bo Tu‡, Lipeng Wu‡, Ying Zhao‡, Xiangyu Liu‡, Lian Li‡, Michael A. McNutt¶, Jingnan Feng‡,
Qihua He?, Yang Yang‡, Haiying Wang‡, Mutsuo Sekiguchi**, and Wei-Guo Zhu‡‡‡2
Fromthe‡KeyLaboratoryofCarcinogenesisandTranslationalResearch(EducationMinistry),DepartmentofBiochemistry
andMolecularBiology,the¶DepartmentofPathology,andthe?BiomedicalTeachingLaboratoryCenter,PekingUniversity
HealthScienceCenter,Beijing100191,China,the§NationalCenterforClinicalLaboratory,BeijingHospital,DongDan,
Beijing100730,China,the**FrontierResearchCenter,FukuokaDentalCollege,Fukuoka814-0193,Japan,andthe
‡‡CancerResearchCenter,PekingUniversityHealthScienceCenter,Beijing 100191,China
S
Proliferating cell nuclear antigen (PCNA) has been demon-
strated to interact with multiple proteins involved in several
metabolic pathways such as DNA replication and repair. How-
ever,therehavebeenfewerreportsaboutwhetherthesePCNA-
bindingproteinsinfluencestabilityofPCNA.Here,weobserved
a physical interaction between PCNA and MutT homolog2
(MTH2), a new member of the MutT-related proteins that
hydrolyzes
(8-oxo-dGTP). In several unstressed human cancer cell lines
andinnormalhumanfibroblastcells,PCNAandMTH2formed
a complex and their mutual binding fragments were confirmed.
It was intriguing that PCNA and MTH2 were dissociated
dependent on acetylation of PCNA, which in turn induced deg-
radation of PCNA in response to UV irradiation, but not in
response to other forms of DNA-damaging stress. To further
explorethelinkbetweendissociationofPCNA-MTH2anddeg-
radation of PCNA, RNAi against MTH2 was performed to
mimicthedissociatedstatusofPCNAtoevaluatechangesinthe
half-lifeofPCNA.KnockdownofMTH2significantlypromoted
degradationofPCNA,suggestingthatthephysiologicalinterac-
tion of PCNA-MTH2 may confer protection from degradation
for PCNA, whereas UV irradiation accelerates PCNA degrada-
tion by inducing dissociation of PCNA-MTH2. Moreover, sec-
ondarytodegradationofPCNA,UV-inducedinhibitionofDNA
synthesis or cell cycle progression was enhanced. Collectively,
our data demonstrate for the first time that PCNA is protected
by this newly identified partner molecule MTH2, which is
related to DNA synthesis and cell cycle progression.
8-oxo-7,8-dihydrodeoxyguanosinetriphosphate
Proliferatingcellnuclearantigen(PCNA)3isamemberofthe
DNA sliding clamp family and consists of a ring-shaped tri-
meric complex (1–3). Three PCNA monomers, each compris-
ing two similar domains, are joined in a head-to-tail arrange-
ment to form a closed ring (4, 5). Because of this unique
structure, PCNA encircles the DNA double helix and slides
freely along it. PCNA was originally characterized as a DNA
polymerase processivity factor and it increases the processivity
of DNA synthesis by interacting with polymerase ? (6, 7). Sub-
sequent studies revealed that PCNA plays an important role in
DNA replication (8, 9). For example, PCNA not only functions
as a protein binding platform to interact with the DNA poly-
merases, flap endonuclease-1 (Fen1) or DNA ligase I (10–12),
butalsocoordinatescomplicatedprocessesinDNAreplication
(2, 13). In addition, PCNA also plays a role in DNA damage
repair (14–17) and cell cycle control (18–20).
Because PCNA is essential for DNA synthesis both in DNA
replication and repair, a dynamic balance between PCNA syn-
thesis and degradation is critical for maintaining normal DNA
synthesis. Up-regulation of PCNA accelerates DNA synthesis
and promotes cell proliferation, such that PCNA is regarded as
a general proliferation marker in tumor development. On the
other hand, degradation of PCNA leads to inhibition of DNA
synthesis (9, 21). In this case, in response to inhibition of DNA
synthesis by PCNA degradation, both cell proliferation and
DNA repair are inhibited, and cells are thus subject to death.
In Escherichia coli, MutT protein encoded by the mutT gene
has 8-oxo-dGTPase activity, and hydrolyzes 8-oxo-dGTP to
8-oxo-dGMP, which is nonutilizable for DNA synthesis, thus
preventing misincorporation of 8-oxo-dGTP into DNA (22).
8-Oxo-dGTP is a product of dGTP oxidation and can be
inserted into opposite dA or dC residues of template DNA at
almost equal efficiencies. As a result, G:C to T:A or T:A to G:C
transversion mutations occur (22–24). In a mutT-deficient
strain, the rate of spontaneous occurrence of A:T to C:G trans-
version increases by 1000-fold compared with that of cells with
wild type mutT (25–27). Therefore, MutT protein is required
forpreventingmutationsandmaintaininghighfidelityofDNA
replication(28).Inaddition,RibAisabackupenzymeforMutT
in E. coli and also plays a role in maintaining high fidelity of
* This study was supported by the National Natural Science Foundation of
China (No. 90919030, No.30425017, 30670417, and 30621002) and Grants
2005CB522403, 2006CB910300, and B07001 from the Ministry of Science
and Technology of China.
□
STheon-lineversionofthisarticle(availableathttp://www.jbc.org)contains
supplemental Figs. S1–S3.
1Both authors contributed equally to this work.
2To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular, Biology, Peking University Health Science Center, 38
Xueyuan Rd., Beijing 100191, China. Tel.:/Fax: 86-1082802235; E-mail:
zhuweiguo@bjmu.edu.cn.
3Theabbreviationsusedare:PCNA,proliferatingcellnuclearantigen;MTH2,
MutT homolog2; 8-oxo-dGTP, 8-oxo-7,8-dihydrodeoxyguanosine triphos-
phate; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluo-
ride; TSA, trichostatin A; ROI, region of interest; FRET, fluorescence reso-
nance energy transfer; PIPES, 1,4-piperazinediethanesulfonic acid; RNAi,
RNA interference; CHX, cycloheximide; TLS, translesion synthesis; RT,
reverse transcription.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 29, pp. 19310–19320, July 17, 2009
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
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DNA replication (29). The MutT homologue MTH1 is the first
MutT-related protein found in mammalian cells (30). The
spontaneous mutation frequency in MTH1-deficient cells
showed an increase of ?2-fold as compared with that in wild
type MTH1 cells (31). Comparing the mutation frequency in
mutT-deficient E. coli cells with that in MTH1-deficient mam-
malian cells suggests that there must be other proteins respon-
sible for preventing occurrence of high numbers of oxidative
damage induced mutations in mammalian cells. By searching
the GenBankTMEST data base, our research group and others
(32) have cloned a new member of MutT-related protein,
MTH2. The increased mutation frequency in mutT-deficient
cells was significantly reduced by overexpression of MTH2
cDNA (32). Therefore, MTH2 may help to ensure cells achieve
accurate DNA synthesis. However, aside from the activity of
8-oxo-dGTPase, the exact mechanism by which MTH2 influ-
ences DNA synthesis has not been explored.
The functions of both PCNA and MTH2 partially overlap in
DNAsynthesis,thuswarrantingexplorationofwhetherMTH2
works together with PCNA to regulate DNA replication or
repair. In this study, we found that MTH2 directly interacts
with PCNA, and this interaction enhances PCNA stability.
However, when cells were exposed to UV light, the interaction
of MTH2 and PCNA was disrupted, and PCNA degradation
was
DNA synthesis was reduced, and
cell cycling was arrested.
accelerated. Consequently,
EXPERIMENTAL PROCEDURES
Cell Culture and UV Irradiation—
Human lung cancer cell lines A549,
H1299, H719 were grown in RPMI
1640 medium supplemented with
10% fetal bovine serum and an
appropriate amount of penicillin/
streptomycin in a 37 °C, 5% CO2,
humidified incubator. Human lung
diploid fibroblast cells (2BS) or
human embryonic kidney 293T
cells (HEK293T) were grown in
Dulbecco’smodified
medium (10% fetal bovine serum).
Cells were plated 24 h prior to UV
treatment. Cells were irradiated at
the indicated doses with a UV lamp
(253.7 nm), and then fresh medium
was changed for incubation at 37 °C
for 3 to 24 h.
Western Blotting—Protein expres-
sion was evaluated by Western blot-
tingaspreviouslydescribed(33).The
antibodies used were anti-PCNA,
-?-tubulin, -histone-H3 (Santa Cruz
Biotechnology),
-phosphotyrosine,
-phosphothreonine (Upstate), anti-
Flag (Sigma), anti-ubiquitin (Santa
Cruz Biotechnology) and anti-
Eagle’s
anti-acetyl-lysine,
-phosphoserine,
MTH2 (a kind gift from Dr. Mutsuo Sekiguchi, mouse
origin).
Co-immunoprecipitation—Co-immunoprecipitation assays
were performed as described previously (34). Briefly, total cell
extract was incubated with antibodies on ice for 1 h. Agarose
A/G was washed three times with Nonidet P-40 buffer (1%
Nonidet P-40, 150 mM NaCl, 50 mM Tris, 0.05% SDS, 1 mM
PMSF,anda1%mixtureofproteaseinhibitors),andthenadded
to the samples, which were then rolled at 4 °C for 1 h. After the
beads were washed three times with Nonidet P-40 buffer, the
pellets were dissolved into 2? SDS loading buffer after centrif-
ugationandboiled5minat100 °C.Theproteinwasanalyzedby
Western blotting with various antibodies.
Triton Extraction—Cells were rinsed twice in cold phos-
phate-buffered saline, then incubated with buffer A (100 mM
NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES at pH 6.8, 1
mM EGTA, 0.2 Triton X-100, 25 mM NaF, 2 mM Na3VO4, 5 mM
PMSF, 2 ?g/ml aprotinin) for 5 min on ice, and the cells were
then scraped and centrifuged. The supernatant was the Tri-
ton-extractable fraction, and the pellet was then lysed in
radioimmune precipitation assay buffer (150 mM NaCl, 1%
Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris at
pH 7.5, 25 mM NaF, 2 mM Na3VO4, 5 mM PMSF, 2 ?g/ml
FIGURE1.PCNAandMTH2formacomplexinvivo.A,proteinswereextractedfromA549,H1299,H719,2BS,
or HEK293T cells for immunoprecipitation using anti-MTH2, followed by Western blotting with anti-PCNA.
B,PCNAwastranslatedinvitrousingpHPCNA15andwasdetectedbyWesternblottingwithanti-PCNA(upper
panel).EndogenousPCNAwasusedasapositivecontrol,andasamplewithoutplasmidwasusedasanegative
control.GSTorGST-MTH2fusionproteinwaspurifiedfrombacteriaandwasdetectedbyWesternblottingwith
anti-GST (middle panel). GST or GST-MTH2 fusion protein was incubated with PCNA (translated in vitro), and
Western blotting was performed to detect interaction with anti-PCNA. C and D, schematic diagrams show
different fragments of GST-MTH2 (upper panel of C) and GST-PCNA (upper panel of D) used in GST pull-down
assays. Both GST-MTH2 and GST-PCNA fusion proteins were purified from bacteria and incubated with total
extractsofA549cells.TheinteractionofPCNAanddifferentfragmentsofGST-MTH2(lowerpanelofC)orMTH2
anddifferentfragmentsofGST-PCNA(lowerpanelofD)wasevaluatedwithWesternblottingusinganti-PCNA
or anti-MTH2, respectively.
PCNAInteractswithMTH2
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aprotinin) and sonicated. After centrifuging, the superna-
tant was Triton-resistant.
Plasmid Construction—GST-MTH2 or GST-PCNA fusion
protein plasmids were generated by inserting MTH2 or PCNA
segments into the vector pGEX-4T-2 with BamHI and XhoI
(New England Biolabs). pECFP-PCNA or pEYFP- MTH2 was
constructed by inserting PCNA or MTH2 segments into the
vector pECFP or pEYFP with XhoI and BamHI. Flag-tagged
PCNA plasmid was generated by inserting wild-type PCNA
into the vector of p3?FLAG- CMV-10. Flag-PCNA mutants
was generated using a site-directed mutagenesis kit (Strat-
agene), following the manufacturer’s directions.
Purification of GST Fusion Proteins and GST Pull-down
Experiments—GST or GST fusion proteins were expressed in
E. coli BL21 (Tiangen Biotechnology, Beijing, China), induced
with 0.5 mM isopropyl-1-thio-?-D-galactopyranoside for 4 h at
30 °C and purified following the protocol from Amersham Bio-
sciences. Equal amounts of GST or GST fusion proteins were
incubated with glutathione-Sepharose 4B beads (Amersham
Biosciences) by rocking at 4 °C for 1 h, and the beads were then
washed three times with TEN buffer (20 mM Tris-HCl, pH 7.4,
0.1 mM EDTA, and 100 mM NaCl). Total cell extracts from
A549cellswereaddedtothebeadsandincubatedbyrockingat
4 °Covernight.ThebeadswerewashedthreetimeswithTENT
buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 7.4, 0.1 mM
EDTA, and 300 mM NaCl), and then dissolved into 2? SDS
loading buffer after centrifugation and boiled 5 min at 100 °C.
After centrifuging, the supernatant was extracted and analyzed
by Western blotting with antibodies to PCNA and MTH2.
Transcription andTranslation
pHPCNA15 (a kind gift from Dr. Moshe Szyf) was linearized
withClaI.LinearpHPCNA15waspurifiedandusedforinvitro
translation employing T7 RNA polymerase (Promega Biotec,
Madison, WI) according to the manufacturer’s instructions. In
vitro translated protein was detected by Western blotting.
Fluorescence Resonance Energy Transfer (FRET)—Both
pECFP-PCNA and pEYFP-MTH2 were transfected into
HEK293Tcells.After48h,cellswereobservedunderaTCSSP2
confocal microscope, and the FRET assay was performed with
TCS SP2 confocal software (Leica, Germany).
Transient Transfection—Plasmids (pECFP-PCNA, pEYFP-
MTH2, p3?FLAG-CMV-PCNA)
HEK293T cells by use of the Qiagen Effectene Transfection kit
(Qiagen, Hilden, Germany) according to the manufacturer’s
instructions.
RNA Interference (RNAi)—siRNA for MTH2 or PCNA
(GeneChem, Shanghai, China) was transfected into A549 cells
byuseofaLipofectamine2000kit(Invitrogen)for48haccord-
in Vitro—Plasmid
weretransfectedinto
FIGURE2.DissociationofPCNAandMTH2inA549cellstreatedwithUV.A–D,A549cellsweretreatedwithUVat46J/m2andthenincubatedfor6horwith
X-raysatdifferentdoses(6Gy,9Gy,12Gy)andthenincubatedfor12h,orwithH2O2(1mMfor12h)orwith5-aza-CdR(1?Mfor72h).Proteinwasextractedfor
immunoprecipitationusinganti-MTH2,followedbyWesternblottingusinganti-PCNAoranti-MTH2.E,A549cellswereirradiatedwithUVatdifferentdosesand
thenincubatedfor4horincubatedfordifferenttimesafterUVirradiationat46J/m2.Proteinwasextractedforimmunoprecipitationusinganti-MTH2,followed
byWesternblottingusinganti-PCNAoranti-MTH2.PCNAbandswerescanned,andrelativebandintensitieswerenormalizedforeachMTH2band.Theband
intensityofCTRwassetas100,andthenumericalvalueoftheintensityofeachbandrepresentsthepercentageofbandintensitywithrespecttothatofCTR.
The representative value in the lower column diagram is an average relative band intensity of PCNA, with standard error from several independent experi-
ments. F and G, both PCNA and MTH2 were detected by Western blotting (F) or RT-PCR (G) in A549 cells irradiated with UV at different doses or for different
incubation times after irradiation. The ?-tubulin loading control is shown in the lower panel. Protein and mRNA bands were scanned, and relative band
intensitieswerenormalizedforeach?-tubulinband.ThelowercolumndiagramsrepresentanaveragerelativebandintensityofPCNAorMTH2withstandard
error from several independent experiments.
PCNAInteractswithMTH2
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ing to the manufacturer’s instructions. To examine the effi-
ciency of siRNA, total protein was extracted for Western blot-
tingusinganti-MTH2or
cccagccaucauuuaucuutt; PCNA siRNA: ccuuggcgcuaguau-
uugatt.
[3H]Thymidine Incorporation Assay—DNA synthesis was
assessed by thymidine incorporation assay. Cells were plated
onto a 6-well plate 24 h prior to experiments. [3H]Thymidine
was added (1 ?Ci/ml) for 12 h, and DNA was precipitated with
cold trichloroacetic acid (50%). The trichloroacetic acid-insol-
uble DNA was placed on a glass microfiber filter (Whatman,
Maidstone, England) and washed twice with cold trichloroace-
tic acid (5%) and then washed once with ethanol or acetone.
Afterairdryingtheglassfilter,itwasputintoavialforcounting
radioactivity (Beckman LS 5000 Liquid Scintillation Counter).
Cell Cycle Analysis—Cell cycle analysis was performed as
described previously (35). UV-irradiated or MTH2-knocked
down A549 cells were harvested and stained with propidium
iodide. The samples were analyzed with flow cytometry (BD
FACS Calibur) using BD CellQuest software.
Immunofluorescence and Confocal Microscopy—A549 cells
were plated on coverslips and grown to 50% confluence. After
thecellswerefixedwith4%paraformaldehydesolutionatroom
temperature for 15 min, they were treated with 0.3% Triton
X-100 at 37 °C for 5 min and blocked with 10% goat serum at
room temperature for 1 h. The cells were then incubated with
1:20dilutionofanti-8-oxo-dGovernightat4 °Candthenwitha
1:100 dilution of Alexa Fluor 488-conjugated goat anti-mouse
IgG (Invitrogen) for 2 h. Images were captured with TCS SP2
confocal microscopy (Leica, Germany). The fluorescence value
was quantified with TCS SP2 confocal software.
anti-PCNA.MTH2siRNA:
RESULTS
PCNA Forms a Complex with MTH2 in Vivo and in Vitro—
ToexploretherelationshipbetweenPCNAandMTH2,wefirst
tested whether PCNA interacts with MTH2 using the co-im-
munoprecipitationassay.EndogenousPCNAwasco-immuno-
precipitatedwithanti-MTH2fromcelllysatesofseveralhuman
non-small cell lung cancer cell lines (A549, H1299, and H719),
human lung diploid fibroblast cells (2BS), and human embry-
onickidney293Tcells(HEK293T).Itwasofinteresttoobserve
that PCNA and MTH2 formed a complex in vivo (Fig. 1A).
Next, to confirm whether the interaction was a direct interac-
tion, a GST pull-down assay was carried out in vitro using bac-
teriallyexpressedproteins.Wild-typePCNAtranslatedinvitro
(Fig. 1B, upper panel) was incubated with a full-length GST-
MTH2 fusion protein (Fig. 1B, middle panel), and Western
blottingwasthenperformedwithanti-PCNA.AsshowninFig.
1B (lower panel), PCNA formed a complex with GST-MTH2
fusion protein in vitro. To further map regions of interaction,
PCNA and MTH2 were divided into three fragments to per-
form GST pull-down in A549 cells. Results showed that the
76–165-amino acid region of MTH2 (Fig. 1C) and 7–100-
aminoacidregionofPCNA(Fig.1D)are the fragments involved
in the interaction of PCNA and MTH2. Although the antibody
ofMTH2wasproducedfrommouseMTH2cDNA,itcaneffec-
tively detect human MTH2 assayed with purified protein in
vitro or by RNAi in vivo (supplemental Fig. S1, A–C). These
results suggested that PCNA physiologically interacts with
MTH2 in cells.
UV Irradiation Induces Dissociation of PCNA and MTH2 in
A549 Cells—To investigate the effect of DNA-damaging
stressesonthePCNA-MTH2complex,A549cellsweretreated
with UV, x-ray, H2O2, or 5-aza-2?-deoxycytidine (5-aza-CdR, a
DNA demethylating agent that causes DNA damage) (35, 36),
and co-immunoprecipitation assays were then performed. It
was a surprise to find that PCNA co-immunoprecipitated with
anti-MTH2 was markedly decreased in UV-treated cells
FIGURE3.FRETanalysisofPCNA-MTH2interaction.AandB,FRETassaywas
performedtodetecttheenergytransferefficiencybetweenPCNAandMTH2
before (A) or after (B) UV treatment. pEYFP-MTH2 and pECFP-PCNA were
transfected into HEK293T cells and visualized by excitation at 514 nm for
EYFP-MTH2(upperpanelofAandB)orat458nmforECFP-PCNA(lowerpanel
of A and B). In the FRET assay, EYFP-MTH2 acted as the acceptor, and ECFP-
PCNA as the donor. In the upper left panel (A and B), the regions chosen for
photobleaching (ROI) are bounded by a white line. C, FRET efficiency was
shown in the column diagram, and the representative value is an average
FRET efficiency of 25 cells with standard error (*, p ? 0.05).
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(Fig. 2A), andthisresultwasdependentonUVdoseorUVtreat-
menttime(Fig.2E).Forexample,comparedwithuntreatedcells,
the interaction of these proteins was decreased 1.3-fold at a
dose of 23 J/m2and 3.7-fold at a dose of 46 J/m2in UV-
irradiated cells as determined by Western blot band inten-
sity measurement (Fig. 2E, left lower column diagram). In
addition, at 3 h after UV irradiation the interaction of these
proteins was decreased 2.3-fold, and at 6 h 5.5-fold (Fig. 2E,
right lower column diagram). It is interesting that there were
no obvious changes in the interaction of these proteins in
cells treated with x-ray, H2O2, or 5-aza-CdR (Fig. 2, B–D),
which shows that UV treatment
may have a unique capacity to
induce a decrease in the interac-
tion of PCNA-MTH2.
To investigate
decrease in interaction between
PCNA and MTH2 is due to dissoci-
ationoftheseproteinsordown-reg-
ulationofindividualproteinexpres-
sion, Western blotting and RT-PCR
were performed to evaluate expres-
sion of PCNA and MTH2 (respec-
tively). Results showed that levels of
both proteins and mRNA were sus-
tained almost
within a period of 6 h after UV irra-
diation (Fig. 2, F and G), indicating
that UV irradiation led to reduced
MTH2/PCNA association and not
to a decrease in the level of either
protein.
In addition, the FRET assay was
performed to confirm the occur-
rence of dissociation of PCNA and
MTH2 in response to UV treat-
ment. HEK293T cells were trans-
fected with plasmids expressing
ECFP-PCNA and EYFP-MTH2. In
this assay, the acceptor photo-
bleaching technique was used to
comparetheemissionofthedonor
fluorophore (ECFP-PCNA) before
and after photobleaching of the
acceptorfluorophore
MTH2) within both ROI 1 and
ROI 2 (ROI, region of interest).
The images on the left panels of
Fig. 3, A and B show the fluores-
cence of EYFP-MTH2 and ECFP-
PCNA before photobleaching. In
unstressedcells,
bleaching of both ROI 1 and ROI 2
of the acceptor (upper right panel,
Fig. 3A), the fluorescence of the
donor in the ROIs was markedly
enhanced (lower right panel, Fig.
3A), demonstrating that FRET had
PCNAand MTH2.
whetherthe
without change
(EYFP-
afterphoto-
occurred
UV-treated cells, after both ROI 1 and ROI 2 of the acceptor
were photobleached (upper right panel, Fig. 3B), the fluores-
cence of the donor in the ROIs showed no obvious change
(lower right panel, Fig. 3B), suggesting that FRET was weak-
ened between these two proteins. Statistically, the energy
transfer efficiency was obviously decreased in UV-irradiated
HEK293T cells as compared with that in untreated cells (Fig.
3C). These data clearly demonstrate that the interaction
between PCNA and MTH2 was disrupted in response to UV
irradiation.
between However, in
FIGURE 4. UV irradiation induces PCNA acetylation in A549 cells. A, A549 cells were irradiated with UV at
46 J/m2and incubated for 6 h. Protein was extracted and then immunoprecipitated using anti-phospho-
tyrosine, phosphoserine, or phosphothreonine, followed by Western blotting using anti-PCNA. Mouse or
rabbit IgG was used as a negative control. PCNA bands were scanned, and relative band intensities were
normalized for each input band. The lower column diagram represents the average relative band intensity of
PCNA with standard error from several independent experiments. B and C, A549 cells were irradiated with UV
at46J/m2andincubatedfor6h(B),orat23J/m2fordifferentincubationtimes(6,12,or24h)(C).Proteinwas
extracted and then immunoprecipitated using anti-PCNA followed by Western blotting using anti-acetyl-
lysine.IgGwasusedasanegativecontrol.Therepresentativevalueofthelowercolumndiagramistheaverage
relative band intensity of PCNA with standard error from several independent experiments. D, epidermal
growthfactor(EGF)wasaddedtohumanA431carcinomacellstoidentifyphosphotyrosineinpp85.A549cells
weretreatedwith5-aza-CdR(0.1?M,48h),x-ray(8-Gy),ordepsipeptide(0.1?M,6h)totestforphosphoserine,
phosphothreonine, or acetyl-lysine of p53, respectively.
PCNAInteractswithMTH2
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UV Induces Acetylation of PCNA—Both acetylation and
phosphorylation have been reported to be related to the
PCNA function (37, 38), which raises a question as to
whetherpost-translational
involved in UV-induced dissociation. To evaluate this ques-
tion, cell lysate from A549 cells was first co-immunoprecipi-
tated with anti-phosphotyrosine, anti-phosphoserine, or
anti-phosphothreonine, and Western blotting was carried
out with anti-PCNA. As shown in Fig. 4A, we did not observe
an obvious change in PCNA phosphorylation at tyrosine,
serine, or threonine sites. However, UV led to significant
acetylation of PCNA (Fig. 4B), and the acetylation was sus-
tained for up to 24 h after UV irradiation (Fig. 4C). In addi-
tion, to test the specificity of these antibodies, A549 cells and
A431 cells were treated with various stimuli. 5-aza-CdR and
x-ray were shown to induce phosphorylation of p53 serine or
modification of PCNAis
phosphorylation of p53 threonine in A549 cells (35). Epider-
mal growth factor (EGF) was shown to stimulate the phos-
photyrosine of pp85 (39). Depsipeptide (a HDAC inhibitor)
was shown to induce the acetylation of p53 lysine (34). As
shown in Fig. 4D, these stimuli significantly induced post-
translational modifications of p53 at these sites which were
tested, demonstrating that acetylation is a unique modifica-
tion of PCNA in response to UV treatment.
To test whether modification of PCNA by acetylation is
relatedtodissociationoftheseproteins,A549cellsweretreated
withTSA(TrichostatinA,aHDACinhibitor)tomimicprotein
acetylation status, and a co-immunoprecipitation assay was
carried out. Results showed that TSA also induced acetylation
of PCNA (Fig. 5A), and dissociation of these proteins followed
(Fig. 5B). However, the level of the two proteins was sustained
in TSA-treated cells (Fig. 5C).
FIGURE5.PCNAacetylationatLys-14isrequiredforthedissociationofPCNAandMTH2.AandB,A549cellsweretreatedwithTSA(1?M,12h),andprotein
was then extracted for immunoprecipitation using anti-PCNA (A) or anti-MTH2 (B), followed by Western blotting using anti-acetyl-lysine or anti-PCNA. The
lower column diagram represents the average relative band intensity of PCNA with standard error from several independent experiments. C, protein was
extractedfromTSA-treatedcells(1?M,12h),andWesternblottingwasperformedusinganti-PCNA,anti-MTH2,oranti-?-tubulin.Thelowercolumnrepresents
the average band intensity of PCNA or MTH2 with standard error from several independent experiments. D and E, wild-type (WT) or K14R Flag-PCNA was
transfected into HEK293T cells for 48 h. Cells were then irradiated with UV at 46 J/m2and incubated for 6 h. Protein was extracted for immunoprecipitation
using anti-Flag, followed by Western blotting using anti-acetyl-lysine (D) or anti-MTH2 (E). The bands representing acetyl-Flag-PCNA (D) or MTH2 (E) were
scanned,andrelativebandintensitieswerenormalizedforeachFlag-PCNAband.Thelowercolumndiagramrepresentstheaveragerelativebandintensityof
acetyl-Flag-PCNA (D) or MTH2 (E) with standard error from several independent experiments.
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Inaddition,toidentifywhichacetylationsiteiscriticalforthe
interaction of these two proteins, several site-directed mutants
of PCNA were established. As PCNA binds to MTH2 via its N
terminus (including Lys-13, Lys-14, Lys-20, Lys-77, Lys-80),
Flag-tagged wild-type and various mutants of PCNA (mutated
Lys-13, Lys-14, Lys-20, Lys-77, or Lys-80) was generated and
transfected in HEK293T cells. However, it was found that UV
irradiation did not induce an obvious increase in PCNAK14R
acetylation as compared with that of wild-type PCNA (Fig.
5D). Consequently, UV could not lead to the dissociation of
PCNAK14Rand MTH2 (Fig. 5E), indicating that acetylation
of PCNA at Lys-14 may play a key role in regulating interac-
tion of MTH2 and PCNA.
Dissociated PCNA Has a Shorter Half-life and Is More
Readily Degraded—PCNA is a highly conserved protein and its
half-life is comparatively long. For example, in NIH/3T3 cells,
the reported half-life of PCNA is 20 h (40). When cyclohexi-
mide (CHX), an inhibitor of protein synthesis, was added into
the medium at 25 ?g/ml for up to 24 h, the expression levels
of PCNA remained unchanged in A549 cells (Fig. 6A, upper
panel). However, in response to UV irradiation, the half-life of
PCNAwasobviouslyshortenedafteralagtime(Fig.6A,middle
panel). For example, the level of PCNA did not obviously
changeintheinitialtimeperiod(?6
h) after UV irradiation. However,
the PCNA level was decreased to
58.7% at 12 h and further decreased
to 21.4% at 24 h as compared with
thelevelofPCNAimmediatelyafter
UV irradiation (0 h) (Fig. 6B).
BecausethePCNAmRNAlevelwas
not changed in response to UV irra-
diation (Fig. 6E), it is likely that UV
irradiation accelerates degradation
of PCNA. To determine whether
there is a link between UV-induced
dissociation of these proteins and
UV-induced degradation of PCNA,
the half-life of PCNA in TSA-
treated or MTH2-knocked down
A549 cells was also evaluated. A
similar result was obtained in the
TSA-treatedcells,inwhichthehalf-
life of PCNA was shortened to
12–24 h (Fig. 6, A, lower panel and
B). Subsequently, knockdown of
MTH2wasusedtosimulatethedis-
sociatedstatus
MTH2. The efficiency of MTH2
RNAi is shown in supplemental Fig.
S1C. The level of PCNA was sus-
tained for up to 24 h after treatment
with CHX in nonspecific RNAi
A549 cells (Fig. 6, C, upper panel
and D). However, the half-life of
PCNA in MTH2-knocked down
cells was shortened to a period of
between 12–24 h (Fig. 6, C, lower
of PCNAand
panel and D), and the mRNA level of PCNA was sustained in
MTH2-knocked down cells as compared with that in nonspe-
cific RNAi cells (Fig. 6F). These data suggest that PCNA disso-
ciatedfromitscomplexwithMTH2hasashorterhalf-lifeandis
more easily degraded.
To further analyze the pathway of PCNA degradation, UV-
irradiated or MTH2-knocked down A549 cells were investi-
gated in the presence of the proteosome inhibitor MG132.
Down-regulation of PCNA induced by UV irradiation or
MTH2-siRNA treatment was reversed in cells in the presence
ofMG132(Fig.7,AandB).Moreover,PCNA-conjugatedubiq-
uitin in UV or MTH2 siRNA-treated cells was increased com-
pared with that in untreated cells (Fig. 7C). These results dem-
onstrate that the degradation of PCNA likely occurs mainly
through a proteosome pathway.
The Degradation of PCNA Is Related to Inhibitory Effects of
UV on DNA Synthesis and Cell Cycle Progression—It is known
that PCNA is involved in many metabolic pathways and is in
particularinvolvedinregulatingDNAsynthesis.Thisledtothe
suppositionthatPCNA-mediatedDNAsynthesismaybeinflu-
enced by MTH2 in response to UV treatment. Thus, an
[3H]TdR incorporation assay was used to evaluate DNA syn-
thesis rate and cell proliferation. As shown in Fig. 8A, a
FIGURE6.Half-lifeofPCNAisshortenedafterdissociationofPCNAandMTH2.AandB,CHXwasaddedinto
untreated,UV-treated,orTSA-treatedA549cellsat25?g/mlfordifferenttimesasindicated.Westernblotting
was performed from whole cell extracts with anti-PCNA (A). The hyphenated line graph shows the change in
PCNA level at different times. The representative value is the average band intensity of PCNA with standard
error from several independent experiments (B). C and D, 24 h after NS-siRNA or MTH2-siRNA was transfected
into A549 cells, CHX was added to the medium at 25 ?g/ml for various lengths of time as indicated. NS-siRNA
wasusedasnegativecontrolfortheRNAiassay.ThehyphenatedlinegraphshowsthechangeinPCNAlevelat
differenttimes.TherepresentativevalueistheaveragebandintensityofPCNAwithstandarderrorfromseveral
independentexperiments(D).EandF,A549cellswereirradiatedwithUVat46J/m2andthenincubatedfor12h
or 24 h (F). NS-siRNA or MTH2-siRNA was transfected into A549 cells for 48 h (G). mRNA was extracted for
RT-PCR.
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significant decrease in [3H]TdR incorporation was detected in
UV-treated cells, and this decrease was reversed by expression
of exogenous PCNA. To further investigate whether knock-
down of MTH2 inhibits DNA synthesis by promoting PCNA
degradation,[3H]TdRwasaddedintothemediumfor12hafter
MTH2-siRNA treatment. Results showed that radioactivity
from[3H]TdRwasobviouslyreducedinMTH2-knockeddown
cells, and this reduction was also reversed by expression of
exogenousPCNA(Fig.8B).Thesedatasuggestthatknockdown
of MTH2 was at least partly associated with inhibition of DNA
synthesis through promoting the degradation of PCNA.
In addition to DNA synthesis, PCNA is also required for
G1-phase progression by interacting with the cyclin D-CDK4
complex(41).Wethereforefurtheranalyzedchangesinthecell
cycle in UV-treated or MTH2-knock down A549 cells. By flow
cytometryanalysis,anobviousG1-phasearrestwasobservedin
both UV-treated A549 cells and MTH2-knocked down A549
cells when compared with the untreated controls (Fig. 8, C–F).
Forexample,afterthetreatmentofUVorRNAiagainstMTH2,
the cells accumulated in G1-phase increased to 64.01% from
55.25% and to 71.09% from 58.78%, respectively. These results
suggested that UV irradiation induced changes in the cell cycle
may be related to PCNA degradation.
Anadditionalquestionofinterest
raised in this study is the fate of
MTH2afterdissociation
PCNAinresponsetoUVtreatment.
To explore the effect of PCNA on
MTH2 activity, A549 cells were
treated with TSA to induce the dis-
sociation of PCNA and MTH2, and
the cellular 8-oxo-dG was then
detectedwith
MTH2, as a 8-oxo-dGTPase, could
degrade 8-oxo-dGTP to 8-oxo-
dGMP, and prevent the misincor-
poration of 8-oxo-dG into DNA.
Therefore, there was a correlation
between the activity of MTH2 and
theamountofcellular8-oxo-dG.As
a positive control, H2O2-treated
cells showed a significant increase
in 8-oxo-dG. Comparably, as a neg-
ative control, N-acetyl-L-cysteine, a
ROS inhibitor, showed an obvious
decrease in 8-oxo-dG. The amount
of 8-oxo-dG was decreased in TSA-
treated cells,
untreated control (supplemental
Fig. S3). However, the difference
between two groups was not signif-
icant by a statistic analysis. It sug-
gests that PCNA may be not a key
factor to regulate the activity of
MTH2.
from
anti-8-oxo-dG.
compared with
DISCUSSION
The data presented here provide
evidence that the newly identified molecule MTH2 is of poten-
tial importance in its functions in association with PCNA. In
particular, our findings demonstrate that the interaction
between PCNA and MTH2 may protect PCNA from degrada-
tion. When these proteins were dissociated in response to UV
irradiation, degradation of PCNA was accelerated, and DNA
synthesis decelerated.
UV irradiation has been reported to induce apoptosis in
many cell lines. For example, human keratinocyte cell line
HaCaT and human leukemia cell line HL-60 were sensitive to
UV-induced apoptosis (42–44). In this study, to avoid apopto-
sisinducedbyUVirradiation,weusedA549cellstoobservethe
PCNA/MTH2 association, as A549 cells were resistant to UV
irradiation-induced apoptosis (45, 46). In addition, LKB1, a
tumor suppressor, is frequently inactivated in non-small cell
lung cancer (47). Therefore, in addition to non-small cell lung
cancer cell lines (A549, H1299, and H719), we also selected
human epithelial cell line 2BS (LKB1-proficient), and human
embryonic kidney 293T cell (LKB1-proficient) to further eval-
uate the interaction of MTH2-PCNA.
Many PCNA-binding proteins, such as p21, polymerase ?,
specific endonuclease Fen1, and XPG contain a consensus
PCNA-interacting motif, referred to as a PIP-box [Q-XX-(h)-
FIGURE7.DegradationofPCNAisproteosome-dependent.AandB,A549cellswereUV-irradiatedat46J/m2
and then incubated for 24 h with DMSO or MG132 at 25 ?M, or NS-siRNA or MTH2-siRNA was transfected into
A549cellsfor48hwithDMSOorMG132at25?M.ProteinswereextractedforWesternblottingwithanti-PCNA.
The lower column diagram represents the average band intensity of PCNA with standard error from several
independentexperiments.C,A549cellsweretreatedwithUV(46J/m2,24h)orMTH2-siRNAinthepresenceof
proteasome inhibitors (25 ?M MG132). The treated cells were then lysed and immunoprecipitated with anti-
PCNA, followed by Western blotting with anti-ubiquitin.
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X-X-(a)-(a)] (48–51). Recently, a novel PCNA binding motif
has been identified, which is called a KA-box [KA-(A/L/I)-(A/
L/Q)-X-X-(L/V)] (52). This motif is also present in several
PCNA-binding proteins, such as polymerase ?, polymerase ?,
and RFC (52). However, the PCNA-binding domain of MTH2
contains neither the PIP-box nor the KA-box motif. Although
MTH2 lacks a consensus motif for PCNA binding, the C-ter-
minal region of MTH2 was demonstrated in this study to bind
to PCNA (Fig. 1C). Consistent with our results, GADD45 has
been reported to bind to PCNA in its C terminus, although
there is no consensus motif in its C-terminal regions (53).
PCNA has previously been found to interact with multiple
different molecules, which are involved in several important
metabolic pathways (2, 54, 55). In addition, the interaction of
GADD45 with PCNA is involved in nucleotide excision repair
(NER) (56, 57). DNA methyltransferase (58, 59), histone
deacetylases (HDAC) (60), and p300 (61–63) also bind to
PCNA to regulate DNA methylation and chromatin remodel-
ing. More importantly, PCNA is able to mediate switching of
different pathways by interacting with different molecules. For
example, PCNA interacts with different DNA polymerases
resulting in a switch from accurate DNA synthesis to error-
prone translesion synthesis (TLS) (64–66). Moreover, monou-
biquitination of PCNA triggers a switching of interactions
betweenPCNAanddifferentpolymerases(67–69).AfterDNA
damage, translesion DNA synthesis (TLS) occurs to prevent
stalling of DNA synthesis, and DNA repair machinery is acti-
vated to maintain DNA integrity. Therefore, after UV irradia-
tion, TLS polymerase or GADD45 binds to PCNA to perform
TLS or nucleotide excision repair (NER). However, PCNA
involved in TLS or NER is chromatin-bound PCNA, whereas
PCNA associated with MTH2 is chromatin-unbound (supple-
mental Fig. S2). Therefore, it seems likely that the dissociation
of MTH2 and PCNA is independent of TLS polymerase or
GADD45.
It has been reported that UV irradiation can induce expres-
sionofPCNA,whereinthemRNAlevelofPCNAwasincreased
by 2-fold over the initial 1–2 h after irradiation as assayed with
Northern blotting (70). However, over a period of up to 4–6
h after UV irradiation, PCNA gradually returned its original
level. Therefore, the up-regulation of PCNA is a relatively
acute response to UV irradiation. This is consistent with our
result that levels of PCNA did not obviously change over the
3–6-h period after UV irradiation (Fig. 2, F and G), although
we did not detect changes in PCNA in the initial 1–2 h after
irradiation.
It has been reported that mammalian cells contain three dif-
ferent PCNA isoforms which differ in their acetylation status,
and these differences are associated with the subcellular local-
ization and functions of these isoforms (37). In this study, we
observed that acetylation of PCNA at Lys-14 was induced by
UV irradiation, and followed the dissociation of PCNA and
MTH2(Fig.5,DandE).Thisthereforesuggeststhatacetylation
of PCNA at Lys-14 is a critical modification, which may bring
FIGURE 8. PCNA degradation inhibits DNA synthesis and cell cycle progression. A and B, after A549 cells were irradiated with UV (46 J/m2), or MTH2 was
knocked down from A549 cells, exogenous PCNA was transfected into the cells. [3H]TdR was then added to the medium at 1 ?Ci/ml for 12 h, and DNA
was precipitated with cold trichloroacetic acid. The radioactivity of three separate experiments was measured, and relative radioactivity per ?g of DNA was
counted.StatisticalanalysiswasperformedbycomparingtheradioactivityofUVtoCTRorMTH2RNAitoNSRNAi(*,p?0.05).TheresultsofRT-PCRinthetop
panels showed the efficiency of expression of exogenous PCNA by transfecting pHPCNA15 into A549 cells (A) and the efficiency of knocking down MTH2 by
transfectingMTH2siRNAintoA549cells(B).C–F,representativeflowcytometryhistogramsat24hafterirradiatingA549cellswithUVat46J/m2,orat48hafter
NS-siRNAorMTH2-siRNAwastransfectedintoA549cells.Theyaxisrepresentsthecellcount,andthexaxisrepresentsDNAcontent.Theseexperimentswere
conducted four times. Differences between groups were statistically analyzed using the unpaired Student’s t test (*, p ? 0.05).
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about a change in PCNA configuration and in turn disrupt the
interaction of MTH2 and PCNA.
AlthoughPCNAfunctionshavebeenwellstudied,themech-
anisms by which its functions are controlled remains to be
investigated. In this study, we observed that the half-life of
PCNA was shorter in UV-treated and MTH2-knocked down
cells (Fig. 6, A–D), and observed that the loss of PCNA was
preventedbytheproteosomeinhibitorMG132(Fig.7,AandB).
However, PCNA did not accumulate in MG132-treated cells as
compared with untreated cells, which is consistent with evi-
dencethathasbeenobtainedbyotherresearchers(38,71).This
lack of PCNA accumulation may result from the fact that
although PCNA degradation is proteosome-dependent, the
degradation level of PCNA is usually very low in nonstressed
cells (72) and an obvious accumulation of PCNA in cells in the
presence of MG132 is thus not observed. Nevertheless, these
results indicated that the dissociation of PCNA and MTH2 is a
trigger for degradation of PCNA and further suggested that
MTH2 forms a complex with PCNA to protect PCNA from
degradation. The region of PCNA cleaved by proteosomes lies
in its N-terminal region (72), which is precisely the MTH2-
binding region of PCNA, and thus suggesting that the binding
of MTH2 to PCNA overlies the N-degron of PCNA. As such,
after the association of PCNA and MTH2 is disrupted by UV,
thedegronofPCNAisexposed,andPCNAdegradationistrig-
gered. Therefore, the association of MTH2 and PCNA stabi-
lizes PCNA, but involves mainly the chromatin-unbound, free
PCNA.
Because PCNA has been used as a proliferation index to
reflect tumor growth because of its high levels in proliferating
cells(55,73–75),reductionofPCNAstabilityoraccelerationof
PCNAdegradationmightofferaneffectivestrategyforregulat-
ingtumorgrowth.UVirradiationisauniversalDNA-damaging
factor, which acts mainly by inducing formation of DNA pho-
toproducts between adjacent pyrimidine bases on the same
strand such as cyclobutane pyrimidine dimers (CPDs) and
(6–4) photoproducts (6–4PPs) (76). UV-induced DNA
photoproducts are the main reason that UV leads to stalled
DNA synthesis. Here our results showed that the expression
ofexogenousPCNAcouldpartlyreversetheinhibitoryeffect
of UV on DNA synthesis (Fig. 8A), suggesting that degrada-
tion of PCNA is one of the reasons that UV particularly
inhibits DNA synthesis.
In addition, UV-induced cell cycle arrest is a dynamic proc-
ess, in which there is a redistribution of cells from G1-phase to
S-phase (77). For instance, UV-irradiated U343 cells initially
accumulated in G1-phase, and reached a peak between 24 and
36h,followedbyanS-phasearrestbetween36and48h.Thisis
consistent with our results wherein an obvious G1-phase arrest
was induced at 24 h after UV irradiation (Fig. 8D).
In conclusion, this study demonstrated that PCNA and
MTH2 form a complex to maintain the stability of PCNA in
unstressed cells. However, UV irradiation induces dissociation
of PCNA and MTH2, which leads to degradation of PCNA.
Therefore, disruption of this specific interaction between
PCNA and MTH2 may prove to be a worthwhile strategy for
inhibition of development of malignant tumors.
Acknowledgments—We thank our colleagues Donglai Wang, Zhixing
Zheng,JingYang,WenZhou,andXiWangfortechnologyusedinthis
work and useful discussions.
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PCNAInteractswithMTH2
19320 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 284•NUMBER 29•JULY 17, 2009
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