a Department of Virology, Institute of Microbiology, University of Oslo, Rikshospitalet, N-0027 Oslo, Norwayb Centre for Molecular Biology and Neuroscience, Institute of Microbiology, University of Oslo, Rikshospitalet, N-0027 Oslo, Norway
c Department of Molecular Biology, Institute of Microbiology, University of Oslo, Rikshospitalet, N-0027 Oslo, Norway
Received 25 August 2005; returned to author for revision 26 September 2005; accepted 3 January 2006Available online 14 February 2006
Keywords: Human cytomegalovirus; DNA base excision repair; Uracil DNA glycosylase; Formamidopyrimidine; Alkylated bases; AP endonuclease; DNAreplication; Genome maintenance; Cell cycle
Introduction
Microbial genomes are constantly challenged by environ-mental agents that induce DNA damage, but the detrimentaleffects of these challenges are normally counteracted bymechanisms of DNA repair. On the other hand, host immuneresponses necessitate mechanisms for fast genome variation anddiversification. Consequently, there is a need for concurrentgenome variability and maintenance, which potentially seemsconflicting. Several mechanisms for genome variation in virusesexist. The low fidelity of viral DNA polymerases may be oneimportant factor for generation of genome variation in DNA-viruses and has been extensively studied in herpes simplex
⁎ Corresponding author. Institute of Microbiology, University of Oslo,Rikshospitalet, N-0027 Oslo, Norway. Fax: +47 23071110.
E-mail address: [email protected](T. Ranneberg-Nilsen).1 Present address: Molecular Imaging Center (MIC), Department of
Biomedicine, University of Bergen, N-5009 Bergen, Norway.
viruses. Song et al. (2004) showed that nucleotide incorporationby the herpes simplex virus type 1 DNA polymerase is lessfaithful than most replicative polymerases. They also found thatmost of the mismatches introduced in DNA were removed bythe DNA polymerase associated 3′- to -5′ exonuclease activityalthough other repair mechanisms may also be involved.However, very little is known to what extent cellular DNArepair mechanisms are active on DNA-viruses. Some DNA-viruses like viruses in the herpes virus group encode for uracilDNA glycosylase (UNG) (reviewed in Chen et al., 2002), whichis a base excision repair enzyme found in most living cells.
DNA damage normally triggers the activity of the cellularDNA repair system. Viruses that induce cellular DNA damageinclude members of the herpes virus group, adenovirus,mumps-virus, measles-virus, rubella-virus, poliovirus, andpapilloma-virus (reviewed in Fortunato et al., 2000). Theregulation of the various DNA repair mechanisms in virus-infected cells has not been well characterized. However, it hasbeen shown that both hepatitis B virus (HBV) and HTLV-I
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infections downregulate nucleotide excision repair (NER). Inhepatitis B virus-infected cells, this downregulation wasattributed the HBV-X protein of both global (Groisman et al.,1999) and transcription-coupled NER (Mathonnet et al., 2004),whereas the HTLV-I tax protein was the active factor in HTLV-I-infected cells (Philpott and Buehring, 1999). It is still an openquestion whether HBV-X protein and HTLV-I tax proteincontribute to the oncogenic potential of these viruses.Adenovirus has been found to downregulate double-strandbreak repair to prevent concatemerization of the viral genome.(Stracker et al., 2002).
It has long been known that HCMV infection is genotoxic tohost cells. It has been shown that HCMV infection inducesaberrations such as chromatid breaks and gaps (AbuBakar et al.,1988). Most of the aberrations seems to be random, however,more recently, it was shown that infection of fibroblasts duringthe S phase of the cell cycle resulted in two site specific breaksat positions 1q42 and 1q21 of chromosome 1 (Fortunato et al.,2000). The underlying mechanisms and biological significanceof these breaks are not known. Furthermore, it has been reportedthat reactive oxygen metabolites are created soon after uptake inHCMV-infected smooth muscle cells (Speir et al., 1998),indicating a potentially genotoxic effect of viral infection. Acrucial question is to what extent cellular as well as viral DNAdamage is repaired during the HCMV infection cycle.
The BER pathway is a multistep process repairing a widerange of endogenous base lesions, including alkylation,oxidation, and deamination products. The damaged bases arerecognized and removed by lesion specific glycosylases whichcleave the N-glycosylic bond between the base and thedeoxyribose. The base less sugar, apurinic/apyrimidinic site(AP site), is subsequently cleaved at the 3′ side by an AP-lyaseactivity, which is associated with bifunctional DNA glycosy-lases or at the 5′ side by an AP endonuclease activity, which isassociated with monofunctional glycosylases. Uracil accumu-lates in DNA by deamination of cytosine or misincorporation ofdUMP resulting in mutagenic U:G mispairs and less harmful U:A base pairs, respectively. In human cells hUNG possess themajor uracil DNA glycosylase activity; however, uracil can beremoved by three other monofunctional DNA glycosylases;hSMUG1 (single-strand-selective monofunctional DNA glyco-sylase), TDG (mismatch-specific thymine DNA glycosylase),and MBD4 (methylated DNA-binding domain protein 4)(reviewed by Scharer and Jiricny, 2001). Five different enzymesare involved in removal of oxidized base residues: hNTH1(Aspinwall et al., 1997), hOGG1 (Bjoras et al., 1997; Aburataniet al., 1997; Arai et al., 1997; Lu et al., 1997; Radicella et al.,1997), NEIL1 (Hazra et al., 2002a), NEIL2 (Hazra et al.,2002b), and NEIL3 (Morland et al., 2002). HOGG1 removes 8-oxoguanine, which is a major mutagenic oxidation product,whereas hNTH1 and NEIL1-3 remove a broad spectrum ofoxidized pyrimidines. All five enzymes remove imidazole-ringfragmented formamidopyrimidine, which is a major cytotoxicoxidation product. AAG is the only DNA glycosylase inmammalian cells for removal of alkylated bases such as 3-methyladenine, 3-methylguanine, and 7-methylguanine (Sam-son et al., 1991).
HCMVencodes for its own uracil DNA glycosylase (HCMV-UNG), and it has been shown that excision of uracil residues isimportant for efficient replication of viral DNA during HCMVinfection (Prichard et al., 1996). The aim of the present work wasto elucidate the BER capacity in a broader context duringHCMV replication in human embryonic fibroblast (HE) cells.We showed that HCMV-infected cells were very proficient forremoval of uracil and 5′ hydrolysis of AP sites (APendonuclease activity) as compared to the mock-infected cells.Furthermore, we showed that the capacity to initiate repair ofalkylated and oxidized base lesions were reduced in HCMV-infected cells. We hypothesize that modulation of BER activitiesmay play an important role in HCMV pathogenesis to ensureefficient replication and genomic variation of viral DNA.
Results
HCMV-infected human primary fibroblast cells were arrestedin the G1/S phase of the cell cycle
Previous studies have shown that HCMV infection inducesarrest of cell growth either in late G1 (Dittmer and Mocarski,1997) or in G2/M (Jault et al., 1995) depending on experimentalconditions (Lu and Shenk, 1996). To characterize the effects ofHCMV on the cell cycle progression in our study, humanprimary fibroblast (HE) cells were synchronized by serumstarvation, subsequently HCMVor mock infected and grown inmedium with 10% serum. Cell cycle progression was monitoredat several time points after infection by flow cytometry. Themock-infected cells followed a normal cell cycle distributionpattern with a cell doubling time of about 24 h. The cellsappeared to cycle until reaching confluence. The DNA contentanalysis of HCMV-infected cells showed an increased amountof DNA per cell throughout the virus replication cycle (Fig. 1).
However, cultivating the cells in the presence of 300 μg/mlof phosphonoformate (PFA) which is a specific inhibitor of viralbut not cellular DNA replication (Dittmer and Mocarski, 1997)abolished the increase in the overall DNA content (Fig. 1). Itthus appears that HCMV-infected cells were arrested in the G1
phase of the cell cycle. This is in the accordance with thefindings of Dittmer and Mocarski (1997) and Lu and Shenk(1996), demonstrating that HCMV infection blocks the S phaseentry of fibroblasts synchronized by serum starvation and thenserum stimulated after infection.
Excision of uracil
Human CMV encodes a gene for a uracil DNA glycosylasewhich is highly conserved in all herpes virus. In order toexamine the level of expression of viral (HCMV-UNG) andendogenous (hUNG) uracil DNA glycosylase in HCMV-infected fibroblasts, nuclear protein extracts were analyzed byWestern blots using peptide specific antibodies that showed nocross-reactivity (data not shown). HCMV-UNG protein (28kDa) was detected 12 hpi and reached a steady-state level 48hpi, whereas hUNG could not be detected 12 hpi but reached apeak of expression 48 hpi (Fig. 2A). These results suggest that a
Fig. 1. Cell cycle analysis of mock- and HCMV-infected synchronized HE cells maintained in 10% serum in the absence (A and B) or presence (C) of 300 μg/ml PFA.The relative DNA content depicted as PI fluorescence intensity and number of cells were determined at 12, 24, 48, 72, and 96 hpi.
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mechanism mediated by HCMV is regulating expression of thehuman uracil glycosylase. Notably, this variation in hUNGexpression is not due to cell cycle regulation since the fibroblastcells remained arrested in G1 phase after HCMV infection.
We next examined the uracil DNA glycosylase activity ofextracts on an oligonucleotide substrate containing a uracil basepairing with adenine (U:A) (Fig. 2B). Extracts of mock-infectedcells showed the highest rate of uracil removal 48 hpi which isin good agreement with the Western analysis and previousresults demonstrating a peak of expression in S phase (Haug etal., 1998). Removal of uracil in HCMV-infected cells increasedfrom 12 to 48 hpi and maintained the same level late in theinfection, supporting the Western blot results (Fig. 2A). It thus
appears that the capacity to remove uracil is significantlyelevated during HCMV infection.
Decreased excision rate of oxidized DNA-bases inHCMV-infected cells
HCMV infection enhances the level of intracellular reactiveoxygen species (ROS) (Speir, 2000). In order to examine ifHCMV infection altered DNA glycosylase activity for repair ofoxidized bases, we analyzed removal of methyl-formamidopyr-imidine (me-faPy). FaPy residues are major cytotoxic purinelesions removed by five different DNA glycosylases involved inrepair of oxidized bases: hNTH1 (Aspinwall et al., 1997),
Fig. 3. Expression of hNTH1 and the excision of me-faPy in HCMV-infectedcells. (A) Western blot analysis of hNTH1 using 15 μg nuclear extracts isolatedat given time points from mock- and HCMV-infected cells. HNTH1 (40 ng) wasused as a positive control for the Western blot experiments. GAPDH was used asa loading control of the same Western blot. (B) Release of me-faPy from [3H]methyl-faPy-poly (dG-dC) DNA by 10 μg nuclear extracts from synchronizedmock- and HCMV-infected cells harvested at given time points. The data aregiven as geometric means of six individual experiments and the error barsrepresent the standard error mean. *P b 0.026 compared with mock-infectedcells.
Fig. 2. Expression of HCMV-UNG and hUNG and uracil excision in HCMV-infected cells. (A)Western blot analysis of HCMV-UNG and hUNG using 10 μgnuclear extracts isolated at given time points from mock- and HCMV-infectedcells. HCMV-UNG (40 ng) and Δ84 hUNG (0.5 ng) were used as positivecontrol for the Western blot experiments. GAPDH was used as a loading controlof the same Western blot. (B) 0.5 μg nuclear extracts harvested at given timepoints from synchronized mock- and HCMV-infected cells were incubated withduplex oligodeoxyribonucleotides containing a single uracil opposite A. Strandcleavage was analyzed by 20% denaturing PAGE and bands detected byPhosphorImaging followed by quantification. 0.l U UNG and 20 ng Nfo wereused as a positive control and no enzyme as a negative control. The data aregiven as geometric means of six individual experiments, and the error barsrepresent the standard error mean. *P b 0.02, **P b 0.004 compared with mock-infected cells.
Fig. 4. Excision of alkylated bases in HCMV-infected cells. Release of alkylatedbases from [3H]-N-methyl-N-nitrosourea treated DNA by 5 μg nuclear extractsfrom synchronized mock and HCMV-infected cells harvested at given timepoints. The data are given as geometric means of six individual experiments andthe error bars represent the standard error mean. *P b 0.005, **P b 0.04compared with mock-infected cells.
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hOGG1 (Bjoras et al., 1997; Aburatani et al., 1997; Arai et al.,1997; Lu et al., 1997; Radicella et al., 1997), NEIL1(Hazra etal., 2002a), NEIL2 (Hazra et al., 2002b), and NEIL3 (Morlandet al., 2002). Excision of me-faPy in HCMV-infected cells wasabout 40% to 50% as compared to mock-infected cells (Fig.3B). This may indicate that the total capacity to repair oxidativedamage is impaired by HCMV. We next examined theexpression level of hNTH1 and hOGG1 by Western blotanalysis of nuclear extracts. The expression level of hOGG1 inmock- and HCMV-infected cells was below the detection level.HNTH1 was constitutively expressed in mock cells, whereasexpression was below the detection level in HCMV-infectedcells at 48–96 hpi (Fig. 3A). This suggests that decreasedexpression of hNTH1 in HCMV-infected cells contribute to thereduced capacity to repair oxidative damage.
Decreased excision of alkyl base residues in HCMV-infectedcells
BER of alkylated bases in DNA is initiated by N3-methyladenine-DNA glycosylase (AAG) (Lefebvre et al.,
1993). Nuclear extracts from mock- and HCMV-infected cellswere tested on DNA treated with N-(3H)-methyl-N-nitrosourea.The amounts of methylpurines formed in such DNA are 65% 7mG, 10% 3 mA, and 0.7% 3 mG (Bjelland et al., 1995). Thealkyl base DNA glycosylase activity in HCMV-infected cellswas significantly reduced (20–50%) compared to mock-infected cells during the entire period of observation (Fig. 4).
Fig. 5. Expression of APE1 and AP endonuclease activity in HCMV-infectedcells. (A) Western blot analysis of APE1 using 1.5 μg nuclear extracts isolated atgiven time points from mock- and HCMV-infected cells. Nfo (40 ng) was usedas positive control for the Western blot experiments. GAPDH was used as aloading control of the same Western blot. (B) 0.5 μg of nuclear extractsharvested at given time points from synchronized mock- and HCMV-infectedHE cells was incubated with duplex oligodeoxyribonucleotides containing asingle THF opposite thymine. Strand cleavage was analyzed by 20% denaturingPAGE and bands detected by PhosphorImaging followed by quantification. 20ng Nfo was used as a positive control and no enzyme as a negative control. Thedata are given as geometric means of six individual experiments and the errorbars represent the standard error mean. *P b 0.05, **P b 0.015 compared withmock-infected cells.
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AP endonuclease activity in HCMV-infected cells
Intact abasic sites generated by monofunctional glycosylasesare further processed by an AP endonuclease to produce single-strand breaks. APE1 is the major AP endonuclease in humancells (Demple et al., 1991). AP endonuclease activity in nuclearextracts of mock- and HCMV-infected cells was measured on aduplex oligonucleotide containing a single synthetic AP site,THF (tetrahydrofurane). HCMV-infected cells showed asignificantly enhanced level of AP endonuclease activity (30–40%) late in the infection cycle as compared to mock-infectedcells (Fig. 5B). Western blot analysis of nuclear extracts with anantibody raised against APE1 showed no significant alterationsin the level of expression at various time points after HCMVinfection as compared to mock-infected cells (Fig. 5A).
Discussion
In the present study, we have investigated the effects ofHCMV infection on the initial steps of BER, which arepossessed by DNA glycosylases cleaving the N-glycosylicbond and subsequent incision of the phosphodiester bond at theabasic site by an AP endonuclease or AP-lyase activity. It wasshown that the DNA glycosylase activity for removal ofoxidized (faPy) and alkylated bases (methylpurines) declined inHCMV-infected cells. In contrast, the capacity to remove uracil
was significantly elevated in infected cells. Notably, thisincrease in uracil DNA glycosylase activity was accompaniedby expression of the HCMV encoded uracil DNA glycosylase.Furthermore, the AP endonuclease activity was enhanced inHCMV-infected cells although the expression level of the majorhuman AP endonuclease was not altered, indicating that eitherHCMV has its own AP endonuclease activity or that APE1 maybe subjected to post-translation modifications that can modulateits activity. These data demonstrate an upregulation of BERactivities that may be involved in viral replication, whereasother BER activities such as glycosylase activities initiatingrepair of alkylated and oxidized bases were downregulated inHCMV-infected cells. It thus appears that modulation of BERactivities may play a role in HCMV infection.
A recent report showed that CD4+ cells from HIV patientswere characterized with increased levels of oxidative DNAdamage accompanied by a marked decline in DNA glycosylaseactivity for repair of oxidative base lesions in CD4+ cells(Aukrust et al., 2005). Notably, CD4+ cells in HIV patientsshowed no significant differences in the capacity to repairalkylated bases. In contrast, in this study, we found that thecapacity to repair both alkylated and oxidative DNA damagedecreased in HCMV-infected cells. Several base lesions arestrongly mutagenic if they remain in DNA during replication. Inparticular, stable oxidation products such as 8-oxoguanine(Cheng et al., 1992) and 5-hydroxycytosine (Hatahet et al.,1993) exhibit strong miscoding properties. It is possible thatincreased oxidative stress combined with significant decline inthe activity for repair of oxidative base lesions in HCMV-infected cells may predispose to elevated mutation frequency(Speir et al., 1998) in the viral genome. These mutations maycontribute to a decrease in sensitivity to antiviral drugs. SeveralHCMV genes are of importance for resistance to antiviral drugsin which the gene products of the HCMV genes UL97(phosphotransferase) and UL54 (DNA polymerase) are targetsfor the most potent anti-HCMV drugs. DNA sequencing ofdrug-resistant HCMV strains worldwide has revealed numerousmutations in both genes (reviewed in Erice, 1999). Moreover,several regions of the HCMV genome exhibit sequence hypervariability including UL73 encoding the glycoprotein gpUL73-gN (Pignatelli et al., 2003), UL74 encoding glycoprotein O(Paterson et al., 2002), UL144 encoding a homologue of theherpes simplex virus entry mediator (Lurain et al., 1999), andUL146 encoding an α-chemokine (Hassan-Walker et al., 2004).These four proteins may play an important role in immuneescape of HCMV. Furthermore, Davis et al. (1999) found thatthe HCMV genome contain several short tandem repeats, microsatellites, that show a high degree of polymorphism, and seemto promote genetic variation in the HCMV-genome. It thusappears that HCMV has developed mechanisms that balance theneed for genomic variation and adaptability on one side and theneed for genomic stability on the other side. Our resultsdemonstrate that reduced capacity to repair mutagenic bases,including several oxidative and methylated base lesions, maycontribute to genome variation in HCMV. We may question ifthe cellular DNA repair machinery gain access to the HCMV-DNA replication compartments. However, recently, it has been
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shown that several cellular DNA repair proteins are recruited tothe herpes simplex virus replication compartments presumablyfor participation in virus DNA replication or repair (Taylor andKnipe, 2004; Wilkinson and Weller, 2004).
Uracil incorporated early during replication of the HCMVgenome must be removed to facilitate transition from early tolate phase replication (Courcelle et al., 2001). Virus replicationis delayed for 48 h in a HCMV-UNG mutant virus, whereas thevirus fails to proceed to the late phase of replication in absenceof both viral and human UNG (Prichard et al., 1996). Our datashowed that expression of cellular UNG is downregulatedduring the initial phase of infection, suggesting that uracilincorporated early during viral replication is removed by viralUNG. However, cellular UNG is temporarily upregulated at alater phase during infection. This could suggest that expressionof cellular UNG may play a role in the switch from the theta tothe rolling circle form of replication that amplifies the viralgenome. We may speculate that the presence of human UNG isessential to recruit cellular proteins involved in replication suchas replication protein A (RPA), proliferating nuclear antigen(PCNA) and DNA polymerase α. It has been shown that inHSV (herpes simplex virus)-infected cells components ofhomologous recombination; RPA, RAD51, and NBS 1, arerecruited to HSV-DNA replication forks by a HSV-1 encodedprotein, UL30 (Wilkinson and Weller, 2004).
In HCMV-infected cells, the enhanced capacity to hydrolyzeabasic sites resulting from base removal support a mechanism inwhich strand breaks generates origins for both the thetamechanism and the highly recombinogenic rolling circlereplication of viral DNA. However, it remains unknownweather this increased 5′ incision activity is encoded by aviral protein, a yet unknown protein or may be the result of apost-translational modification to APE1. It is becoming evidentthat different post-translational modifications as phosphoryla-tion and acetylation seem to play a role in determining thefunctional activity of the APE1 protein (Pines et al., 2005).
In summary, we have demonstrated that some of the initialsteps in BER are downregulated during HCMV replication inhuman fibroblasts. The lowered BER activity in HCMV-infected cells may contribute to increased genomic variation ofthe virus. Increased excision rate of uracil and enhanced APendonuclease activity in HCMV-infected cells may form gaps inviral DNA required for efficient viral DNA synthesis at latetimes in viral replication, according to a model for HCMV-DNAreplication by Courcelle et al. (2001).
Materials and methods
Cells
Human embryonic fibroblasts (HE) cells were obtained fromthe National Institute of Public Health (Folkehelsa, Oslo,Norway). HE cells were maintained in minimal essentialmedium (MEM) (Gibco, Life Technologies Ltd., Scotland,UK) supplemented with 2% endotoxin-free and mycoplasmafree fetal calf serum (FCS), L-glutamine (0.3 mg/ml),gentamicin (40 μg/ml), amphotericin B (Fungizone) (2.5 μg/
ml), and penicillin G (6 μg/ml). MEM with 10% FCS was usedfor propagation of the cells. The HE cells were routinelyscreened for mycoplasma with DNA staining with bisbenzi-mides (Behring Werke AG, Marburg, Germany).
Virus preparation
Highly purified cytomegalovirus (HCMV) stocks propagat-ed in low passage human embryonic fibroblast (HE) cells wereused. Low-passage HE cells and the laboratory type strainAD169 (American Type Culture Collection (ATCC, Rockville,MD) were propagated at low virus to cell ratios to minimizegeneration of defective particles. The supernatant was harvestedwhen an extensive cytopathogenic effect was evident andcentrifuged briefly at 400 × g to remove cell debris. Thesupernatant was then centrifuged at 48000 × g for 90 min. Thepellet was placed on a gradient of potassium tartrate-glycerolprepared in PBS, centrifuged at 125,000 × g for 60 min, and theband with the purified virus was then harvested. Medium wasadded, and the purified virions were pelleted at 48,000 × g for90 min. The virus was resuspended in medium, titrated in low-passage HE cells, and frozen in aliquots at −70 °C (HCMVvirus titer 1 × 107– 5 × 107 pfu/ml). Mock preparations weremedium supplemented with the serum and antibiotics of thesame batches as the virus preparations. They were aliquot andfrozen at −70 °C in the same way as purified virus.
Cell cycle synchronization and infections
After trypsination 104 cells/cm2 were allowed to grow for48 h before washing the cells 3 times with Hank's balancedsalt solution (HBSS, Gibco, Paisley, UK). The cells (80%confluence) were synchronized in Go phase (data not shown)by serum starvation (0.2% serum) for 72 h prior to infections.The cells were then infected with HCMV at a multiplicity ofinfection (MOI) of 5 plaque forming units (pfu) per cell. Virusadsorption was allowed for 1.5 h in serum-free medium. Afterinfection the cells were restimulated with 10% FCS andfurther incubated. The cells were harvested at 12 h, 48 h, 72 h,and 96 h post-infection.
Only experiments where more than 95% of the cells wereinfected were accepted. The infection was assessed at 24 hpi byan immunocytochemical method employing E13 monoclonalantibodies specific for IE1 and 2 antigens (Seralab, Sussex,UK).
Cell cycle analysis by flow cytometry
Mock- and HCMV-infected cells and HCMV-infected cellsgrown in the presence of 300 μg/ml of PFA (phosphonoformate,P-6801 (Sigma), harvested at the indicated time points, werewashed in PBS, pelleted, resuspended in PBS, and then fixed inice-cold 70% ethanol for at least 30 min at −20 °C. Afterstaining of the cells with propidium iodide (50 μg/ml) andRNaseA treatment (100 μg/ml), the cells were subjected to flowcytometric analysis (BD LSR flow cytometer (Becton Dick-inson, San Jose, CA, USA)), and the results were analyzed with
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the CellQuest software (Becton Dickinson, San Jose, CA,USA).
Preparation of nuclear protein extracts
Nuclear extracts were made by plasmolysis by a modifiedprocedure as described (Bjelland et al., 1995). Briefly, cellsfrom 162-cm2 flasks were harvested, washed twice in PBS,pelleted by centrifugation (1000 × g, 4 min), then lysed with100 μl Milli Q water and left on ice for 10 min before adding100 μl 0.85% NaCl. Cells were then pelleted by centrifugation(5000 × g for 10 min) and plasmolyzed on ice for 10 min with25 μl 84% sucrose in 10 mM EGTA/40 mM Tris–HCl, pH 8.0,100 μl lysis buffer (50 mM MOPS, pH 7.5, 1 mM EDTA, 100mM KCl and 10 mM β-mercaptoethanol and proteaseinhibitors; 0.5 mM PMSF, 1 μg/ml each of pepstatin A andleupeptin), was added before the pellet was snap frozen 3 timesin ethanol/dry ice followed by centrifugation (20,000 × g, 10min). The nuclear proteins were collected in the supernatant andkept in aliquots at −20 °C until use. BioRad protein assay wasused to measure the protein concentrations using bovine serumalbumin (BSA) as standard (Bradford, 1976).
DNA substrates
N-[3H]methyl-N′-nitrosourea (MNU) (18 Ci/mmol) wasused to prepare poly (dG-dC) DNA containing faPy residues(5000 dpm/μg DNA) as described (Boiteux et al., 1984) andalkyl base DNA (8000 dpm/μg) as described (Bjoras et al.,1995). Uracil (U, a 25-mer oligonucleotide containing a U atposition 13; 5′GCTCATGCGCAG[U]CAGCCGTACTCG3′)and tetrahydrofuran (THF, a 36-mer oligonucleotide contain-ing tetrahydrofuran at position 20; 5′GCTGTTGAGATCCAGTTCG[THF]AGTAACCCACTCGTGC3′) containing oligo-nucleotides were used as substrates in enzymatic analysis.The oligonucleotides were 32P-end labeled at the 5′-endusing [γ-32P] ATP (3000 Ci/mmol; Amersham Biosciences)and T4 polynucleotide kinases (New England Biolabs). Afterseparation of the oligonucleotide from the residual γ-32P-ATPusing NAP-5 columns, the purified oligonucleotides werehybridized with complementary oligonucleotides. Uraciloligonucleotide was paired with adenine (A) in the comple-mentary strand, whereas THF was paired with thymine (T),respectively.
Enzymes
Purified E. coli Nfo was used as positive control in the uracilDNA glycosylase and in the AP endonuclease assays. Nfo waspurified from E. coli using the His6 Ni-affinity purificationSystem (Qiagen) as recommended by the manufacturers.Human NTH1, purified as described (Eide et al., 2001), wasused as a positive control in Western blot experiments. ThehUNG recombinant protein, Δ84 UNG, coding for the catalyticdomain of the hUNG protein (lacking the N-terminal 84 aminoacids (27 kDa, 230 amino acids) was used as a positive controlfor Western blot and was a kind gift from Dr. Hans Krokan
(Norwegian University of Science and Technology, Trondheim,Norway). Uracil DNA glycosylase was purchased from MBIFermentas.
Enzymatic reactions
Unless otherwise stated, all enzyme activities were assayedin a reaction buffer containing 70-mM MOPS pH 7.5, 1 mMEDTA, 5% glycerol, and 1 mM DTT, and the mixtures wereincubated at 37 °C for 30 min. FaPy-DNA and alkyl base DNAglycosylase activities were measured in a total volume of 50 μlcontaining 0.4 μg me-faPy and 0.2 μg alkylated bases,respectively. The amount of released [3H] me-faPy or [3H]alkylated bases was determined from the amount of ethanol-soluble radioactivity in the supernatant after ethanol precipita-tion of the substrate (Eide et al., 1996). Uracil and THF assayscontained 50 fmol of 32P-labeled oligonucleotides. Samples (10μl) were incubated for 30 min at 37 °C. The cleavage productswere analyzed on 20% denaturing DNA sequencing gels andPhosphorImaging (Molecular Dynamics).
Western blot analysis
For Western blot analysis, nuclear extracts were subjected to15% SDS-PAGE and probed with either a rabbit polyclonalanti-HCMV-UNG raised against a synthetic peptide comprisingthe amino acids 119–134 of HCMV-UNG, a rabbit polyclonalagainst the hUNG catalytic domain (PU101) kindly provided byDr. Hans Krokan (Norwegian University of Science andTechnology, Trondheim, Norway) a rabbit polyclonal anti-hNTH1 (Luna et al., 2000) or a monoclonal antibody toGAPDH (cat #4300, Ambion) as a loading control. Proteinbands were detected using a phosphatase conjugated anti-rabbitor anti-mouse IgG secondary antibody (Promega) and color-metric visualized with Alkaline Phosphatase substrate (Gibco,Life Technologies).
Statistics
GraphPad Prism® version 4.03 (GraphPad Software, SanDiego CA) was used. Data are presented as mean values andstandard error of the mean (SEM) of six individual experiments.Paired t test was used for all figures. A two-tailed P value 0.05was considered significant.
Acknowledgment
This work was supported by The Norwegian Cancer Society(grant 88186/004).
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