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[Cancer Research 63, 5799-5807, September 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Base Excision Repair Deficiency Caused by Polymerase ß Haploinsufficiency

Accelerated DNA Damage and Increased Mutational Response to Carcinogens1

Diane C. Cabelof2, ZhongMao Guo2, Julian J. Raffoul, Robert W. Sobol3, Samuel H. Wilson, Arlan Richardson and Ahmad R. Heydari4

Department of Nutrition and Food Science, Wayne State University, Detroit, Michigan, 48202 [D. C. C., J. J. R., A. R. H.]; Department of Physiology, University of Texas Health Science Center, San Antonio, Texas, 78284 [Z. M. G., A. R.]; Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709 [R. W. S., S. H. W.]; and Geriatric Research, Education and Clinical Center, Audie L. Murphy Division, South Texas Veterans Health Care System, San Antonio, Texas 78284 [A. R.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The base excision repair pathway (BER) is believed to maintain genomic integrity by repairing DNA damage arising spontaneously or induced by oxidizing and alkylating agents. To establish the role of DNA polymerase ß (ß-pol) in BER and ß-pol-dependent BER in maintaining genomic stability, we have measured the impact of a gene-targeted disruption in the ß-pol gene on DNA repair capacity and on in vivo sensitivity to carcinogens. We have extensively phenotyped the DNA ß-pol heterozygous (ß-pol+/-) mouse as expressing ~50% less ß-pol mRNA and protein and as exhibiting an equivalent reduction in the specific activity of ß-pol. We measured BER activity by in vitro G:U mismatch and 8-OHG:C repair and find that there is a significant reduction in the ability of extracts from ß-pol+/- mice to repair these types of DNA damage. In vivo, the ß-pol+/- mice sustain higher levels of DNA single-strand breaks as well as increased chromosomal aberrations as compared with ß-pol+/+ littermates. Additionally, we show that reduction in ß-pol expression and BER activity results in increased mutagenicity of dimethyl sulfate as evidenced by a 2-fold increase in LacI mutation frequency. Importantly, the ß-pol+/- mice do not exhibit increased sensitivity to DNA damage induced by N-nitroso-N-ethylurea, ionizing radiation, or UV radiation, which induce damage processed by alternative repair pathways, demonstrating that this model is specifically a BER-deficient model.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The BER5 pathway repairs damage to DNA that arises spontaneously as a result of alkylation, oxidation, and deamination events and is also responsible for repairing small, nonhelix distorting lesions that may be induced by chemical carcinogens. The BER pathway is also responsible for the repair of abasic sites, which may arise spontaneously as a function of temperature fluctuations or which may arise as intermediates in the DNA repair process. In line with the predominance of these types of DNA damage, it is estimated that the BER pathway repairs up to 1,000,000 nucleotides/cell/day (1) . The abundance of damage induced endogenously each day that must be repaired by the BER pathway illustrates the importance of this pathway in maintaining genomic integrity and inefficiency in this pathway could have significant detrimental effects on genomic stability. One method of determining the relative importance of any biological process is to remove it from the system and measure the effects of this loss. The ability to target specific genes for disruption allows researchers to measure the phenotypic impact of the loss of a gene product.

The BER pathway functions by a series of well-coordinated enzymatic events, and several gene products may be targeted in an attempt to disrupt the overall efficiency of the system. BER is initiated by glycosylase-mediated removal of a damaged base or by the presence of an abasic site. Glycosylases have specificity to particular base lesions and may function as monofunctional proteins (remove damaged base only) or bifunctional proteins (remove damaged base and incise DNA backbone). Homozygous deletions in several of the DNA glycosylases have been generated, and as a group, they show mild phenotypes with increased susceptibility to oxidative stress (OGG1 knockout; Ref. 2 ) and alkylation stress (APNGknockout; Ref. 3 ). In the case where a monofunctional glycosylase initiates BER, the subsequent step will be incision of the DNA backbone by an endonuclease, creating appropriate 3'- and 5'-termini for polymerase insertion of an undamaged base. The endonuclease responsible for this step is Ape1 (also called Hap1, Ref1, and Apex), and a homozygous deletion in this gene is embryonic lethal. The heterozygous mouse survives and has been described by Meira et al. (4) . In the case where BER is initiated by a bifunctional glycosylase, the DNA backbone is already incised, but the termini generated do not support polymerase insertion of a new base. The backbone requires additional processing to generate a 3'-OH group, and this trimming appears to be accomplished by Ape1 (reviewed in Ref. 5 ). Nucleotide insertion is completed by DNA ß-pol. ß-Pol performs the polymerization step as well as the rate-limiting step of dRP removal in monofunctional glycosylase-initiated BER (6) and is essential for polymerization in bifunctional glycosylase-initiated BER. A third subpathway described for BER is termed long-patch BER because the insertion of two to eight nucleotides, as opposed to just one nucleotide, occurs during the repair process. In vitro, this long-patch pathway is described as being instrumental in the processing of modified abasic sites that are resistant to dRP excision by ß-pol. The relative importance of ß-pol in this minor subpathway appears to be reduced in comparison to its role in the two major subpathways because ß-pol null cells are able to support long-patch repair (7) , however, it has been demonstrated that ß-pol conducts repair synthesis in long-patch repair (8, 9, 10) . Full elucidation of the long-patch pathway remains to be completed. Completion of BER is accomplished upon ligation by DNA Ligase I (11) or DNA Ligase III/XRCC1. Homozygous deletion of XRCC1, which acts as a scaffolding protein for both ligase III and ß-pol (12, 13, 14) , is embryonic lethal (15) .

This article addresses the phenotypes observed in response to heterozygous gene-targeted disruption of ß-pol. Homozygous deletions in the ß-pol gene are embryonic lethal, but the heterozygous mice developed by Rajewsky’s lab (16) survive and are fertile. The embryonic lethality observed for three of the BER genes (Ape1, ß-pol, and XRCC1) suggests a critical role for these proteins in particular and for BER in general. Although the use of a homozygous-knockout model provides an ideal picture of the roles of these proteins, the use of the heterozygous models is made necessary by their embryonic lethality. Although genetic attempts can and should be made to rescue these animals, the use of the heterozygous mice is exciting because they may provide us with models relevant to the human population. We provide evidence that half the gene dosage of ß-pol results in genomic instability. Although this haploinsufficiency does not affect the spontaneous development of tumors in young animals, we demonstrate that it does increase the mutagenic response to carcinogen exposure. In line with the suggestion that DNA repair genes may act as caretaker genes (17) , BER haploinsufficiency may modify cancer risk in individuals.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Experiments were performed in male-specific pathogen-free mice in accordance with the "NIH Guidelines for the Use and Care of Laboratory Animals," and the animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio, the Subcommittee for Animal Studies at Audie L. Murphy Memorial Veterans Hospital, and the Wayne State University Animal Investigation Committee. Mice were maintained on a 12-h light/dark cycle and were fed a standard mouse lab chow and water ad libitum. Mice were sacrificed at appropriate ages by cervical dislocation. Organs were flash frozen in liquid nitrogen and stored at -70°C for later enzyme studies.

Mice heterozygous for the DNA ß-pol gene (ß-pol+/-) were created in Rajewsky’s laboratory by deletion of the promoter and the first exon of the ß-pol gene (16) . The animals appear to be normal and are fertile; there is no retardation in food intake, weight gain, or growth rate. The genotype of the mice was determined by Southern blot analysis as described by Sobol et al. (18) . As shown in Fig. 1ACitation , the embryonic stem cells with a null mutation in ß-pol were generated by a type I deletion of the promoter and the first exon of the ß-pol gene. During this process a BamHI site was introduced in the site of the deletion. Thus, upon digestion of genomic DNA with BamHI, the ß-pol+/+ gene exhibits a 10-kb band, whereas the mutated ß-pol+/- gene exhibits a 3-kb band. Fig. 1BCitation shows that DNA isolated from the tails of ß-pol+/- mice give both the 10- and 3-kb bands in contrast to DNA isolated from C57BL/6 mice, which shows only the 10-kb band.



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  		Fig. 1. Genotyping and expression of ß-pol gene in ß-pol+/+ and ß-pol+/- mice. A, restriction map for the 5'-portion of the mouse wild-type ß-pol locus and the type I deletion. Upon digestion of genomic DNA with BamHI, the wild-type ß-pol gene exhibits a 10-kb band, whereas the mutated ß-pol gene exhibits a 3-kb band. B, Southern blot analysis of BamHI-digested genomic DNA isolated from tail sections of a litter of nine. The genomic DNA isolated from the ß-pol+/+ mice exhibits a 10-kb band, whereas the genomic DNA from the ß-pol+/- mice exhibits a 10-kb band (wild-type ß-pol locus) and a 3-kb band (type I-deleted ß-pol gene). C, sample of autoradiographs of Northern blots of ß-pol and GAPDH mRNA levels in brain, liver, spleen, and testes of ß-pol+/- mice and normal, littermates. Poly(A)+ RNA was isolated from various tissues of mice using a kit from Invitrogen (FastTrack 2.0 mRNA Isolation Kit), and the level of ß-pol in 5 µg of the poly(A)+RNA was determined by Northern blot hybridization using a cRNA probe to ß-pol. As a control, the levels of GAPDH mRNA in the RNA samples were also determined. D, the relative level of ß-pol mRNA in various tissues of ß-pol+/- mice and normal littermate mice was quantified using a Bio-Rad Molecular Imager, and the data were normalized based on the GAPDH mRNA levels; the data are shown in the graph and expressed as percentage of mRNA levels in the tissue of the ß-pol+/+ mice. Each point represents mean ± SE for data obtained from three separate experiments in which RNA samples were pooled from two mice for each experiment. *, value significantly different from ß-pol+/+ mice at P < 0.01.

 
The ß-pol+/- and ß-pol+/+ littermates were maintained under barrier conditions in a temperature-controlled environment and fed ad libitum with a mouse/rat chow (Harlan Teklad, Madison, WI). To study the effect of ß-pol haploinsufficiency on glycosylase function, ß-pol+/- and ß-pol+/+ mice at 12 months of age were injected i.p. with 2-NP (100 mg/kg body weight) dissolved in olive oil. Mice were sacrificed by cervical dislocation 3, 6, 24, and 48 h after injection. Mouse tissues were collected immediately for measurement of 2-NP-induced DNA lesions using HPLC. To study the effect of ß-pol haploinsufficiency on mutation frequency, the ß-pol+/- mice were crossbred to LacI-transgenic mice (Stratagene, La Jolla, CA). These cross-breedings produced mice heterozygous for ß-pol gene and hemizygous for LacI transgene (ß-pol+/-/LacITg+/0) and mice hemizygous for LacI transgene (LacITg+/0). The DMS (CAS no. 77-78-1; Sigma, St. Louis, MO) experiments were carried out in young (4–6 months) male heterozygous for ß-pol gene and hemizygous for LacI transgene (ß-pol+/-/LacITg+/0; Stratagene). Animals were injected with 10, 30 and a total dose of 50 mg DMS/kg body weight administered as a single 30 mg/kg i.p. injection followed by two weekly injections of 10 mg/kg each, or with vehicle only. An expression period of two weeks was allowed before animals were sacrificed and liver DNA was collected for mutational analysis as described below.

Isolation of Crude Nuclear Extract.
All tissues were handled on ice or at 4°C during isolation of nuclear proteins. Tissues were homogenized in a buffer [10 mM HEPES (pH 8.0), 1.5m MgCl2, 10 mM NaCl, 10 mM NaS2O5, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and 1 µg/ml pepstatin A] and then centrifuged for 10 min at 10,000 x g at 4°C. The pellet was mixed with 1.5 volumes of homogenization buffer plus 1 M NaCl, homogenized again, then centrifuged at 100,000 x g at 4°C. The nuclear proteins were precipitated by addition of a 40% (NH)4SO4, and stirring for 30 min. Precipitated materials were collected by centrifugation at 15,000 x g for 20 min at 4°C. The pellet was dissolved in a minimal volume of dialysis buffer [20 mM Tris (pH 8.0), 100 mM KCl, 10 mM NaS2O5, 0.1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml pepstatin A] and dialyzed against the buffer for 1 h at 4°C using Slide-A-Lyzer cassettes (Pierce Chemical Company). Insoluble materials were removed by centrifugation at 12,000 x g for 10 min at 4°C. The supernatant was aliquoted and stored at -70°C for use in repair assay, gap-filling assay, and Western blot analysis. Protein concentration of nuclear extracts was determined according to Bradford (19) .

Expression Analysis.
Northern analysis was performed in various tissues. The poly(A)+ RNA was isolated using a kit from Invitrogen (FastTrack 2.0 mRNA Isolation Kit). The levels of ß-pol mRNA were measured by cRNA/RNA hybridization using {alpha}-[32P] cRNA probe to ß-pol (20) . The levels of ß-pol mRNA were expressed relative to the 18 s rRNA and GAPDH mRNA levels.

Western analysis was performed in various tissues. Nuclear extracts from tissues of ß-pol+/+ and ß-pol+/- animals were subjected to SDS-PAGE and transferred to nitrocellulose using a Bio-Rad semidry transfer apparatus according to the manufacturer’s protocol. SDS-PAGE was conducted in duplicate; one gel was stained with Coomassie blue to ensure that the same quantity of protein was loaded onto the gels, and the other gel was used to quantify ß-pol protein levels. Western analysis was accomplished using affinity purified monoclonal antisera developed against mouse ß-pol or monoclonal antisera developed against mouse p53 (pAb240). The bands were detected by ChemiImager after incubation in SuperSignal Chemiluminescent Substrate luminol/enhancer and SuperSignal chemiluminescent Substrate stable peroxide solution (Pierce Chemical Company). Intensity of the bands was quantified using a Molecular Dynamics densitometer, and the data are expressed as the integrated intensity of the band/µg protein loaded.

Gap-filling Assay/ß-Pol Activity.
A 5-nucleotide-gapped oligonucleotide duplex (upper strand, 5'-GCTTGC ATGCCTGCAGGTGTACGT—-GATCCCCG GGTACCGAGC-3', the 5-nucleotide gap indicated by the dashes; lower strand, 3'-CGAACGTACGGACGTCCACATGCAATTGCCTAGGGGCCCATGGCTCG-5') is 5' end-labeled and incubated at 37°C for 30 min with a DNA synthesis reaction buffer [4x buffer: 200 mM Tris (pH 8.0), 40 mM MgCl2, 80 mM NaCl, 10% glycerol; 1.25 mM dATP, dCTP, dGTP, dTTP, and 100 mM DTT; crude nuclear extract, extraction procedure described above; 5 µg/ml aphidicolin). DNA was resolved on a 12% polyacrylamide sequencing gel and visualized and quantified using a Bio-Rad Molecular Imager. The data are expressed as machine counts/µg of protein. DNA resolving at 24 bases indicates an absence of gap filling, whereas DNA resolving at 47 bases indicates that gap-filling synthesis has occurred.

DNA Repair Assay.
An oligonucleotide (upper strand, 5'-ATATACCGCGGUCGGCCGATCAAGCTTA TTdd-3'; lower strand, 3'-ddTATATGGCGCCGGCCGGCTAGTTCGAATAA-5') flanked with dideoxy ends containing a G:U mismatch was incubated with nuclear extract from tissues of ß-pol+/+ and ß-pol+/- mice in a reaction mixture containing 100 mM Tris (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.1 mM ATP, 0.5 mM NAD, 5 mM diTris-phosphocreatine, 10 units of Creatine phosphokinase, 20 µM dATP, dTTP, and dGTP with 2 µM dCTP plus 10 µCi of radiolabeled dCTP. The mixtures were incubated for 10 min at 37°C, and the DNA was extracted with phenol-chloroform and precipitated. Reactions were also completed with aphidicolin to block DNA polymerases {alpha} and {delta} and with neutralizing antibody to ß-pol. The purified oligonucleotides were separated on a 12% polyacrylamide denaturing sequencing gel. Repair of the synthetic oligonucleotide results in the incorporation of radiolabeled deoxynucleotide triphosphate, which is visualized and quantified using a Molecular Imager System (Bio-Rad, Hercules, CA). The data are expressed as machine counts/µg of protein.

Mutation Analysis.
The mutation analysis was performed as described by Walter et al. (21) . In brief, genomic DNA was isolated from mouse livers using the RecoverEase DNA isolation kit per manufacturer protocol. The LacI transgene was recovered from genomic DNA by the Transpack in vitro packaging kit from Stratagene, according to manufacturer protocol. Packaged phage was mixed with Escherichia coli SCS-8 cells from Stratagene and plated on NZY agar assay trays containing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside. The number of mutant lacI genes was divided by the total number of plaque-forming units to determine the mutation frequency.

Comet Assay for ssDNA Breaks.
The comet assay was carried out under dim light to prevent artifactual DNA damage. The livers were minced into small pieces (1–2 mm3) in ice-cold HBSS (HBSS, Ca2+ and Mg2+ free) that also contained 20 mM EDTA and 10% DMSO. The liver tissue was washed three times with the HBSS to remove most of the RBCs and then minced into very small pieces. The minced tissue was allowed to sit undisturbed in a tube for 5 min, and cells in suspension were obtained. The cells were collected as a pellet by centrifugation and resuspended in the HBSS at a concentration of 1 x 105 cells/ml. The comet assay was used to measure DNA strand breaks as described by Singh et al. (22) , with minor modifications. Briefly, a layer of 75 µl of 1% standard agarose in PBS was layered on a microscope slide and overlayered with a mixture of 10 µl of cell suspension and 75 µl of 0.5% low-melting agarose in PBS. Cells were lysed by immersing the slide in 2.5 M NaCl, 0.1 M EDTA, 0.1 M Tris-HCl (pH 10), 10% DMSO, 1% Triton X-100 at 4°C for 2 h. The slides were then immersed in an electrophoresis buffer: [300 mM NaOH and 1 mM EDTA (pH 12.1)] for 20 min to allow DNA unwinding. The electrophoresis was carried out at 300 mA and ~20 V for 30 min. The slides were then treated with 0.4 M Tris(hydroxymethyl) aminomethane (pH 7.5) and stained with Sybr green (Molecular Probes, Inc., Eugene, OR). Slides were viewed using a fluorescence microscope (Nikon, 100-W Hg lamp). The fluorescence image of a single cell was analyzed using a Komet 4.0 SCG image analysis system (Integrated Laboratory System, Inc., Research Triangle Park, NC). Fifteen cells were randomly analyzed from each slide, and three slides were examined for each liver sample. The tail moment was used as a measure of the degree of damage to DNA, which was calculated from the tail length (distance from center of head to tail end) multiplied by the percent tail DNA.

Determination of 8-OHdG.
Genomic DNA was isolated with a NaI DNA extractor kit (Wako Chemicals, Inc., Richmond, VA) as described by Hamilton et al. (24) . This technique, which uses NaI instead of phenol, minimizes DNA oxidation that occurs during DNA isolation (23 , 24) . Thirty µg of DNA were hydrolyzed using nuclease P1 and calf alkaline phosphatase. The levels of both 8-OHdG and 2dG in the DNA hydrolysates were quantified using a HPLC-EC detection system with a polar mobile phase [100 mM sodium acetate, 5% methanol (pH 5.2)] as described previously (25 , 26) . The level of DNA oxidation is expressed as the ratio of 8-OHdG to 2dG.

Chromosomal Aberration Assay.
Bone marrow cells were flushed from the femurs of the mouse into 10 ml of RPMI 1604 supplemented with 0.2 µg/ml Colcemid and 10% fetal bovine serum. After being cultured at 37°C for 60 min, the cells were pelleted and suspended in hypotonic solution (0.075 M KCl) for 20 min, centrifuged, and fixed with 3:1 methanol-acetic acid. After two additional washes with fixative, cells were resuspended in a small volume of the fixative, and a thin layer of the cell suspension was applied to a microscope slide. The slide was then stained uniformly with Wright’s Giemsa without banding. Chromosomal aberrations were scored using a Zeiss Axioskop photomicroscope.

Statistical Analysis.
Statistical significance between means was determined using ANOVA followed by the Fisher’s least significant difference test where appropriate (27) . Data for the Comet assay are reported as mean ± SE. The differences between control and treated and/or between ß-pol+/+ and ß-pol+/- mice were analyzed by two-factor ANOVA followed by Sidak’s multiple comparison test. P < 0.01 was considered statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We have extensively characterized the ß-pol+/- mice with respect to tissue-specific expression of ß-pol. We have measured by Northern blot hybridization levels of ß-pol mRNA in poly(A)+RNA isolated from the brain, liver, spleen, and testes of ß-pol+/- mice and their wild-type littermates (Fig. 1C)Citation . The data in Fig. 1DCitation show that we consistently observed a 40–50% decrease in the levels of ß-pol mRNA in tissues of the ß-pol+/- mice. Subsequently, we measured the levels of ß-pol protein in tissues of ß-pol+/+ and ß-pol+/- mice by Western blot analysis. These experiments were important because we could not assume that the differences in ß-pol mRNA would accurately affect differences in ß-pol protein levels. We observe a corresponding 50–60% decrease in ß-pol protein in ß-pol+/- mice in all tissues studied (Fig. 2, A and B)Citation . To confirm that changes in the expression of ß-pol (i.e., changes in mRNA and protein levels) give rise to changes in ß-pol activity, we measured ß-pol activity in the brain, liver, and testes of ß-pol+/- and ß-pol+/+ mice using a gap-filling reaction (28) . The data in Fig. 2, C and DCitation , show that the ß-pol activity in the tissues from ß-pol+/- mice was ~50% less than that observed in tissues from ß-pol+/+ mice. Thus, ß-pol levels and activity are reduced in tissues of the ß-pol+/- mice as would be expected based on the gene dosage.



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  		Fig. 2. Analysis of protein level and ß-pol enzymatic activity in ß-pol+/+ and ß-pol+/- mice. A, samples of autoradiographs of Western blots of ß-pol protein levels in brain, liver, and testes of ß-pol+/- mice and normal littermates. Crude nuclear extracts were isolated from various tissues of mice (brain, liver, and testes), and the levels of ß-pol protein in 50 µg of nuclear extract were determined by Western blot analysis using an enhanced chemiluminescence detection kit. B, the relative level of ß-pol protein in various tissues of ß-pol+/- mice and normal littermates was quantified using an {alpha} Innotech MultiImage system, and the data were normalized based on the amount of protein loaded on each gel. The data are shown in the graph and expressed as percentage of ß-pol protein levels in the tissue of the ß-pol+/+ mice. Each point represents mean ± SE for data obtained from three separate experiments in which nuclear extracts were pooled from two mice for each experiment. *, value significantly different from ß-pol+/+ mice at P < 0.01. C, autoradiographs of sequencing gel electrophoresis of primer extension activity of ß-pol. A synthetic oligonucleotide (51 bases long) was used as template in which the sequence of the 6-nucleotide gap between a 24-base prime and a 21-base 5'-phosphorylated downstream primer was designed as a substrate for the gap-filling reaction of ß-pol enzyme. The reaction was carried out with 32P-labeled primer for 30 min at 37°C in the presence of 50 µg of nuclear extract isolated from various tissues (brain, liver, and testes) of ß-pol+/- mice and normal littermates. D, the relative level of ß-pol activity in various tissues of ß-pol+/- and ß-pol+/+ littermate mice were quantified using a Bio-Rad Molecular Imager, and the data were normalized based on the amount of protein used in each reaction. The data are shown in the graph and expressed as percentage of ß-pol activity in the tissue of the ß-pol+/+ mice. Each point represents mean ± SE for data obtained from three separate experiments in which nuclear extracts were pooled from three mice for each experiment. (+Ab, Cont.), addition of antibody against ß-pol to inhibit ß-pol polymerase activity and to serve as a control; *, value significantly different from ß-pol+/+ mice at P < 0.01.

 
The experiments described above establish a deficiency in the expression of ß-pol gene in ß-pol+/- mice, however, ß-pol is only one enzyme in a cascade of enzymes involved in the BER pathway. Therefore, it was necessary to determine whether the changes in ß-pol expression resulted in a decreased ability to repair DNA damage. As such, we measured the ability of tissues from ß-pol+/+ and ß-pol+/- animals to repair a uracil-containing synthetic oligonucleotide. In this assay, incorporation of radiolabeled dCTP indicates repair activity. BER activity was determined in brain, liver, spleen, and testes and is significantly reduced in tissues from the ß-pol+/- mice (Fig. 3)Citation . The addition of neutralizing antibody to ß-pol completely blocked repair (far right lane, Fig. 3ACitation ), whereas the addition of aphidicolin had no effect (data not shown), demonstrating the critical role of ß-pol in these repair reactions. Using a modification of this assay, we have also measured the ability of nuclear extracts from ß-pol+/+ and ß-pol+/- animals to repair a synthetic oligonucleotide containing an 8-OHdG residue and find that extracts from ß-pol+/- mice show reduced (30–40%) ability to repair this damage (data not shown).



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  		Fig. 3. BER assay in ß-pol+/+ and ß-pol+/- mice. A, an autoradiograph of a sequencing gel indicating G:U mismatch repair activity as visualized by the appearance of a 30-base fragment. Samples were pooled from extracts obtained from three to four animals in each group. B, the relative levels of BER in various tissues of mice were quantified using a Bio-Rad Molecular Imager, and the data were normalized based on the amount of protein used in each reaction. The data are shown in the graph and expressed as percentage of BER activity in the tissue of the ß-pol+/+ mice. Values represent an average (±SE) for data obtained from at least three animals in each group. (+Ab, Cont.), addition of antibody against ß-pol to inhibit repair activity and to serve as a control; *, value significantly different from ß-pol+/+ mice at P < 0.01.

 
To determine whether the initial steps in BER are properly functioning in the ß-pol+/- mice, both ß-pol+/+ and ß-pol+/- animals were injected with 100 mg/kg body weight of 2-NP. 2-NP induces 8-OHdG, aminated guanines, which are prone to oxidation, hydrazinohypoxanthine (29, 30, 31, 32) , and DNA strand breaks that correspond tightly to 8-OHdG induction (32 , 33) . 8-OHdG was measured in various tissues of ß-pol+/+ and ß-pol+/- mice before and after treatment with 2-NP. As shown in Fig. 4Citation , basal levels of 8-OHdG in both groups of mice were the same. In response to 2-NP, both groups of animals showed significant induction in 8-OHdG levels 3 h after 2-NP injection in all of the tissues studied; however, no significant differences were observed between ß-pol+/- and ß-pol+/+ mice. Because induction of 8-OHdG is greatest in liver, we have further investigated whether this effect might change over time. As such, we have measured 8-OHdG levels from liver tissue of ß-pol+/- and ß-pol+/+ mice at 3, 6, 24, and 48 h after 2-NP exposure (Fig. 5)Citation and find that the removal of 8-OHdG remains the same in both groups of animals over time. These results demonstrate that a 50% reduction in ß-pol/BER activity does not alter the removal of 8-OHdG from DNA, demonstrating that glycosylase-initiated removal of 8-OHdG is fully functional in ß-pol+/- mice.



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  		Fig. 4. The induction of 8-OhdG levels in various tissues of ß-pol+/+ and ß-pol+/- mice treated with 2-NP. Twelve-month-old mice were injected i.p. with 2-NP (100 mg/kg). The amount of 8-OHdG in the DNA hydrolysate was measured by HPLC and EC detector as described in methodology. The data are expressed as values relative to the amount of 2dG detected by UV absorbance at 290 nm. Values represent an average (±SE) for data obtained from at least three animals in each group.

 


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  		Fig. 5. The induction of 8-OhdG levels and ssDNA break in liver tissue of ß-pol+/+ and ß-pol+/- mice treated with 2-NP. Twelve-month-old mice were injected i.p. with 2-NP (100 mg/kg). The amount of 8-OHdG in the DNA hydrolysate was measured by HPLC and EC detector as described in methodology 3, 6, 24, and 48 h after treatment. The data are expressed as values relative to the amount of 2dG detected by UV absorbance at 290 nm (bar graph). The induction of DNA strand breaks by 2-NP was determined by quantifying the relative amount and spatial distribution of DNA that migrated out of the nuclei of hepatocytes obtained form control and 2-NP-treated ß-pol+/+ and ß-pol+/- mice (line graph). Values represent an average (±SE) for data obtained from at least three animals in each group. *, value significantly different from ß-pol+/- mice at P < 0.01.

 
DNA ß-pol removes the dRP residue after AP endonuclease cleaves DNA at AP sites, and ß-pol inserts a new nucleotide in the resulting gap. Until these ß-pol-mediated steps are completed, a break in one strand of the DNA will persist. Therefore, a decrease in ß-pol activity could result in an increase in DNA strand breaks. We measured the level of DNA strand breaks in liver cells obtained from ß-pol+/+ and ß-pol+/- mice using the comet assay under alkaline conditions. Fig. 6ACitation shows photographic images of how the DNA damage is classified in this study. Class I nuclei possess no or few DNA strand breaks as the fluorescence image is confined to the nuclei; displayed tail moments range from 0 to 5 µm. In cells with DNA strand breaks, a fluorescence tail becomes visible because small DNA fragments have migrated out of the nuclei. Class II nuclei display tail moments between 5 and 15 µm. Class III and IV nuclei both display tail moments > 15 µm but can be distinguished visually. Class III nuclei have a pinhead separated from a puffy tail that is associated with the DNA fragmentation occurring in apoptosis. Class IV nuclei look like class II nuclei but with a larger tail moment and represent cells with high DNA strand breaks perhaps associated with necrosis. As shown in Fig. 6BCitation , the percentage of cells with class I nuclei are significantly lower in the ß-pol+/- mice compared with the ß-pol+/+ mice, demonstrating that the ß-pol+/+ mice sustain lower levels of DNA damage. In contrast, the percentage of cells with classes II, III, and IV nuclei is greater in the ß-pol+/- mice, demonstrating that the ß-pol+/- animals sustain greater levels of DNA damage in the form of strand breaks. Additionally, we have shown that strand breaks arising as a result of exposure to an oxidizing agent (2-NP) are greater and persist for longer in the ß-pol+/- mice (Fig. 5)Citation . Because 8-OHdG is induced in response to 2-NP but efficiently removed in both ß-pol+/- and ß-pol+/+ mice, we suggest that the persistence of strand breaks in the ß-pol+/- animals represent DNA repair intermediates caused by stalling at the ß-pol-mediated steps and a subsequent inability to ligate the strand break.



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  		Fig. 6. Single-strand breaks in liver cells of ß-pol+/+ and ß-pol+/- mice. Mouse liver cells were prepared and analyzed using the comet assay as described in "Materials and Methods." A, examples of comet images of cells with class I, II, III, and IV nuclei are shown. B, one hundred fifty cells were analyzed for each liver sample and classified as belonging to one of four class comets. The percentage of cells in class I and the percentage of cells in class II, III, and IV were calculated. Values represent an average (±SE) for data obtained from at least six mice in each group. The differences between ß-pol+/- and ß-pol+/+ mice were analyzed by an unpaired t test (nonparametric test). *, value significantly different from ß-pol+/+ mice at P < 0.05.

 
Ionizing radiation produces oxidative DNA damage and strand breaks. In this study, we expose the whole-body of ß-pol+/- and ß-pol+/+ mice to 8.5 Gy of {gamma}-radiation. This dose has been demonstrated to kill 50% of mice within 30 days. Our study showed that ~50% of mice died 30 days after irradiation; there was no significant difference in survival between ß-pol+/- and ß-pol+/+ mice (Fig. 7)Citation . Thus, the ß-pol+/- mice show no increased sensitivity to the killing effects of ionizing radiation, an effect that may mask any sensitivity the BER-deficient animals may have to the oxidative damage induced by ionizing radiation.



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  		Fig. 7. Survival of WT and ß-pol+/- mice exposed to {gamma}-radiation. Mice (10 ß-pol+/+ and 10 ß-pol+/-) at 6 months of age were exposed to 8.5 Gy of {gamma}-radiation using a 137cesium {gamma}-source (dose rate, 1.05 Gy/min). The mice were observed for 35 days after exposure, and changes in mortality were recorded daily.

 
ß-Pol-null cells derived from embryonic fibroblasts exhibit increased sensitivity to monofunctional DNA-alkylating agents (18) and increased mutation frequency in response to methyl methanesulfonate (34) , suggesting that ß-pol is required for repairing alkylation damage. In this study, we treated ß-pol+/-/LacI+/0 and ß-pol+/+/LacI+/0 mice with DMS, an alkylating agent, and measured the mutation frequency in the LacI transgene from liver DNA of these mice. As shown in Fig. 8Citation , the basal level of mutation frequency in ß-pol+/-/LacI+/0 and ß-pol+/+/LacI+/0 mice is similar. However, in response to every dose of DMS tested, the mutation frequency is significantly higher in ß-pol+/- as compared with ß-pol+/+ animals. At the 30 mg/kg dose, an 80% increase in mutation frequency in ß-pol+/- compared with ß-pol+/+ mice is observed, whereas at the total dose of 50 mg/kg, a 2.5-fold increase in mutation frequency in response to DMS is observed in the ß-pol+/- mice. DMS is a fairly well-tolerated carcinogen, as evidenced by the lack of mutational response observed in wild-type animals at the lowest doses. In contrast, the ß-pol+/- animals exhibit an increase in mutation frequency in response to DMS even at the lowest doses, demonstrating that levels of DNA damage that are well tolerated in ß-pol+/+ animals are poorly tolerated in ß-pol+/- animals. This establishes a phenotype of reduced DNA damage tolerance in this model of BER deficiency. That is, these animals exhibit an in vivo phenotype of BER deficiency in that carcinogens induce more damage when the damage induced is characteristic of damage repaired by the BER pathway.



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  		Fig. 8. The effect of a reduction in BER capacity on the spontaneous and chemically induced mutation frequencies in the lacI transgenes in ß-pol+/+ and ß-pol+/- mice. Animals were treated with DMS or olive oil (carrier) as described previously (20) . Animals were sacrificed 14 days after the last dose of the treatment. The mutation frequency of the lacI transgene in liver tissues of control and DMS-treated ß-pol+/+ and ß-pol+/- mice was determined. Values represent an average (±SE) for data obtained from at least three animals in each group. *, significantly different (P < 0.01); **, P < 0.05.

 
To determine whether these animals are specifically BER deficient and do not exhibit deficiencies in alternative repair pathways, we have measured sensitivity of these animals to DNA-damaging agents that induce a spectrum of damage distinct from those repaired by BER. O6-alkyl lesions, for example, are repaired by a direct reversal mechanism and are highly mutagenic. In response to ENU (150 mg/kg body weight), which induces ~25-fold greater levels of the O6 lesions than DMS, we observe no increased mutagenicity in spleenocytes from ß-pol+/- animals as compared with ß-pol+/+ animals (Fig. 9)Citation . In response to UV exposure, which induces a high level of cyclobutane prymidine dimers repaired by the nucleotide excision repair pathway, we also observe no increased sensitivity in the ß-pol+/+ compared with ß-pol+/- mice (data not shown). These data suggest that heterozygocity in the ß-pol gene specifically effects ß-pol-dependent BER activity, whereas alternative pathways, e.g., nucleotide excision repair and direct reversal, remain intact.



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  		Fig. 9. The effect of a reduction in BER capacity on the spontaneous and chemically induced mutation frequencies in the lacZ transgene in ß-pol+/+ and ß-pol+/- mice. Animals were treated with ENU or olive oil (carrier). Animals were sacrificed 14 days after the treatment. The mutation frequency of the lacZ transgene in spleenocytes of control and ENU-treated ß-pol+/+ and ß-pol+/- mice was determined. Values represent an average (±SE) for data obtained from at least three animals in each group.

 
DNA strand breaks can conceivably give rise to chromosomal aberrations through a variety of mechanisms. Because ß-pol+/- mice exhibit an increased percentage of cells with DNA strand breaks, we hypothesized that they might exhibit increased levels of chromosomal aberrations. We have measured the chromosomal aberrations in the mouse bone marrow cells and observed an increased centromere separation in the bone marrow cells obtained from ß-pol+/- mice (Table 1)Citation . Increased single-strand break and chromosomal aberrations with time in ß-pol+/- mice demonstrate that the ß-pol+/- animals are more sensitive to an accumulation of DNA damage, suggesting an increased sensitivity to endogenous damage. Additionally, we find that from 3 to 12 months of age, the ß-pol+/+ mice exhibit no difference in p53 protein levels in liver tissue, while over the same time frame the ß-pol+/-, mice exhibit a significant increase in p53 protein (Fig. 10)Citation . Because the p53 protein is stabilized in response to DNA damage, this increase in the ß-pol+/- animals may provide additional evidence that ß-pol haploinsufficiency results in an accelerated accumulation of DNA damage.


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  		Table 1 Chromosomal aberrations in bone marrow cells of 12-month-old ß-pol+/+ and ß-pol+/- mice

 


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  		Fig. 10. The induction of p53 levels in liver of tissue of 12-month-old ß-pol+/- mice. The level of p53 protein in 20 µg of nuclear extract was determined by Western blot analysis using an antibody against p53 protein and a SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical Company, Rockford, IL). Samples were pooled from extracts obtained from three to four animals in each group. The relative level of p53 protein in liver tissue was quantified using an {alpha} Innotech MultiImage system, and the data were normalized based on the amount of protein loaded on each gel. Values represent an average (±SE) for data obtained from at least three animals in each group. *, value significantly different from ß-pol+/+ mice at P < 0.01.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Establishing a viable model of BER deficiency will provide researchers with a valuable tool for assessing the importance of this pathway in maintenance of genomic stability and may provide a useful model for aging and cancer research. The data presented in this study establish the ß-pol+/- mouse as a BER-deficient animal, both in terms of in vitro and in vivo repair capacity. The observed reductions in ß-pol mRNA, protein, and activity along with the reduced ability of extracts from ß-pol-deficient mouse tissues to repair a damaged oligonucleotide clearly define the ß-pol+/- mouse as a BER-deficient model. In light of emerging data suggesting roles for other polymerases with ß-pol-like activity in BER (35) , it is critical that we have observed a reduction in repair activity caused by heterozygous deletion of ß-pol. This allows us to conclude that other polymerases are not compensating for the reduction in ß-pol. Additionally, this reduction in BER activity is not affected by the addition of aphidicolin, which blocks DNA polymerases {alpha} and {delta}, but is completely inhibited by the addition of neutralizing antibody to ß-pol, demonstrating that the reduction in ß-pol is responsible for the reduction in BER activity.

A question that remains is whether a 50% reduction in ß-pol has an in vivo effect. Although the in vitro data are compelling, it is possible that there are in vivo mechanisms of adaptation to ß-pol deficiency that are immeasurable in our in vitro system. Although the glycosylate-mediated steps of BER function properly in the ß-pol+/- mice, we suggest that the inability to additionally process the resulting abasic sites and/or incised DNA backbones are affected by ß-pol haploinsufficiency. As such, we have demonstrated that basal ssDNA breaks increase with time in the ß-pol+/- mice and have additionally shown that ssDNA breaks are greater in ß-pol+/- mice after exposure to the DNA-damaging agent 2NP. In BER, completion of repair, i.e., ligation, is not possible until polymerization and/or dRP removal have occurred. As such, if the amount of the polymerase responsible for insertion of nucleotides in the BER pathway is limited, as it is in this ß-pol+/- mouse, then the increase in single-strand breaks we observe may be a function of incomplete repair intermediates that could subsequently generate double-stranded DNA breaks as these repair intermediates can act as Topoisomerase II poisons (36) . Accordingly, we also observe a significantly higher level of chromosomal aberrations in the ß-pol+/- mice (centromere separations). It may be that the persistence of repair intermediates caused by a limiting amount of ß-pol is responsible for the increase in chromosomal aberrations. In agreement, Sobol et al. (37) have demonstrated that ß-pol-null fibroblasts accumulate chromosomal aberrations in response to MMS. Horton et al. (38) demonstrate that ß-pol-/- cells are hypersensitive to 5-hydroxymethyl-2'deoxyuridine (hmdUrd), suggesting that removal of damaged bases in the absence of the subsequent repair steps (ß-pol in this case) is cytotoxic. Additionally, we show that single-strand breaks persist for longer in the ß-pol+/- mice in response to oxidative stress, which may be expected to induce greater levels of chromosomal aberrations. In agreement with accelerated accumulation of DNA damage in the ß-pol+/- mice, we observe a significant increase in p53 protein levels over time that is not observed in the wild-type mice. It is interesting to suggest that the lack of ß-pol/BER may directly affect the regulation of p53. In light of evidence suggesting a tight correlation between p53 levels and BER activity, the relevance of this relationship should be further investigated.

To determine whether the ß-pol+/- mice show increased sensitivity to carcinogens, we have measured sensitivity to a variety of DNA-damaging agents. These sensitivity data allow us to determine the importance of a ß-pol-dependent pathway in the repair of damages induced by these agents. By measuring sensitivity to both DMS and ENU, we showed that the ß-pol+/- mice are specifically more sensitive to DMS, which induces lesions known to be processed by BER (primarily N7-meG; Ref. 39 ) while showing no significant difference in sensitivity to ENU as ENU induces lesions not processed by BER (primarily O6-alkylguanines and alkylphosphates; Ref. 40 ). The primary lesion induced by DMS is N7-meG (80% of lesions) and only 0.3% O6-meG (39) . Lindahl (in T. A. Kunkel; Ref. 41 ) estimates the number of N7-meG lesions at 4000/day. Although normally well tolerated, the inability to adequately process these lesions is detrimental as evidenced by Elder et al. (3) who show that homozygous deletion of APNG results in a 3–4-fold increase in the mutagenicity of MMS (similar damage spectrum to DMS). Thus, the inability to remove N7-meG significantly increases the mutagenicity of a normally well-tolerated lesion. Accordingly, we show that doses of DMS well tolerated in the wild-type animals are mutagenic in the ß-pol+/- animals. It is unlikely that this increased mutagenicity is a function of O6-meG lesions, typically the most likely carcinogenic alkyl lesion (40) because ENU, which induces 8% O6-alkylGuanine (42) —a 25-fold increase above the amount of this lesion induced by DMS—causes no sensitivity in the ß-pol+/- mice. Additionally, it is well understood that the O6-alkyl lesions are repaired by direct reversal and not by a BER pathway. Thus, we are confident that the induction in mutation frequency we see in response to DMS cannot be attributable to the O6-alkylGuanine lesion and is instead attributable to the inability to process BER substrates such as N7-meG and N3-meAdenine.

With respect to the sensitivity of these mice to oxidative stress, we measured survival after exposure to IR and found no difference in survival between wild-type and ß-pol+/- mice. Because 70% of radiation-induced damages result from IR-induced free radicals (43) , we expected to see sensitivity in the ß-pol+/- mice. However, cell killing effects of IR are a function of DNA double-strand breaks (44) , not oxidized bases, and this effect may have masked any sensitivity to IR. Still, this lack of sensitivity to IR caused us to revisit whether a ß-pol-dependent pathway was responsible for the repair of oxidized bases. It is clear that oxidized bases are processed by a variety of mechanisms: 8-OHdG and thymine glycol lesions existing on the transcribed strand of actively transcribed genes are repaired by a CSB-dependent mechanism (45) ; the XPA nucleotide repair-deficient mouse is sensitive to oxidative stress (46) ; and mismatch repair pathways have been implicated in the processing of these lesions (47) . In addition, we show here that the ß-pol+/- mice are more sensitive than the wild-type mice to oxidative stress induced by 2-NP as evidenced by higher and more persistent levels of DNA single-strand breaks, with a significant difference between the two groups being maintained with time. We propose that in response to induction in 8OHdG, a bifunctional glycosylase removes the damaged base and creates a strand incision, as expected. However, subsequent repair synthesis is delayed in the ß-pol+/- mice as a result of limiting amounts of ß-pol, resulting in persistence of single-strand breaks, increasing the likelihood of chromosomal damage occurring as a result of oxidative stress in the ß-pol+/- animals.

Emerging data point to a complicated and coordinated sequence of events in the completion of BER involving a variety of different protein:protein interactions, a growing list of accessory proteins (PARP, p53, PCNA) and divergent repair pathways for the removal of similar types of DNA damage (i.e., oxidative damage). The biological relevance of the effect of one individual protein or any one single type of DNA damage is difficult to interpret in isolation from the whole and provides at best another part of the story in the maintenance of genomic integrity. The data presented in this work support an important role for a ß-pol-dependent pathway in the protection of genomic stability through maintenance of adequate BER capacity. The implication that a deficiency in BER increases the damaging effects of normally well-tolerated base lesions and increases sensitivity to endogenous damage supports a role for BER in both aging and cancer. The concept that the DNA damage threshold can be affected by BER capacity is supported by our previous work demonstrating a connection between the age-related loss of ß-pol/BER and DMS sensitivity (20) , by the demonstration that ß-pol-null cells are sensitive to much lower concentrations of MMS than their ß-pol wild-type cells (38) , and by the data presented in this article.

An inability to tolerate carcinogen exposure resulting from ß-pol haploinsufficiency has important human health implications. For example, many somatic mutations at Arg283 of ß-pol have been described in human tumors [Expressed Sequence Tags (EST) database, National Center for Biotechnology Information]. As Arg283 is known to be required for full DNA synthesis activity (48) , its mutation on one chromosome would render a cell haploinsufficient for ß-pol and, therefore, BER deficient. Similarly, DNA variations or polymorphisms in the ß-pol gene occur in the human population. Although many are as yet uncharacterized with respect to functionality, the results reported here indicate that polymorphisms altering enzyme function on one chromosome could impact the cell through haploinsufficiency. Because we see no spontaneous tumors in this animal model early in life, we can define ß-pol as a low penetrance gene, requiring interaction with a high penetrance mutation, high penetrance environmental exposure, or perhaps aging for cancer to develop. This is supported by studies showing that haploinsufficiency in other low penetrance DNA repair genes results in increased tumorigenesis upon interaction with high penetrance mutations (49 , 50) . In this study, we demonstrate that ß-pol haploinsufficiency increases the mutagenic response to carcinogen exposure. Thus, the results described here have important implications for future research on understanding the role of base excision repair in individual susceptibility to genotoxic stress.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants 1R21-DK62256 (to A. R. H.), 1PO1-AG14674 (to A. R.), and P01-AG19316 (to A. R.), the American Institute for Cancer Research (to A. R. H.), the San Antonio Nathan Shock Aging Center (1P30-AG13319), and the American Institute for Cancer Research (to A. R. H.). Back

2 D. C. C. and Z. M. G. contributed equally to this work. Back

3 Present address: Hillman Cancer Center, University of Pittsburgh Cancer Institute, Research Pavilion, Suite 2.6, 5117 Centre Avenue, Pittsburgh, PA 15213-1863. Back

4 To whom requests for reprints should be addressed, at Department of Nutrition and Food Science, 3009 Science Hall, Wayne State University, Detroit, MI 48202. Phone: (313) 577-2427; Fax: (313) 577-8616; E-mail: Ahmad.Heydari{at}wayne.edu Back

5 The abbreviations used are: BER, base excision repair; ß-pol, polymerase ß; dRP, deoxyribose phosphate; 2-NP, 2-nitropropane; HPLC, high-performance liquid chromatography; DMS, dimethyl sulfate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 8-OHdG, 8-hydroxy-2-deoxyguanosine; 2dG, 2-deoxyguanosine; EC, electrochemical; ENU, ethylnitrosourea; ssDNA, single-stranded DNA; N7-meG, N7-meGuanine; IR, infrared. Back

Received 4/ 5/03. Revised 6/ 6/03. Accepted 7/ 2/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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