Imbalanced Base Excision Repair in Response to Folate Deficiency Is Accelerated by Polymerase β Haploinsufficiency
2004; Elsevier BV; Volume: 279; Issue: 35 Linguagem: Inglês
10.1074/jbc.m405185200
ISSN1083-351X
AutoresDiane C. Cabelof, Julian J. Raffoul, Jun Nakamura, Diksha Kapoor, Hala Abdalla, Ahmad R. Heydari,
Tópico(s)Trace Elements in Health
ResumoThe mechanism by which folate deficiency influences carcinogenesis is not well established, but a phenotype of DNA strand breaks, mutations, and chromosomal instability suggests an inability to repair DNA damage. To elucidate the mechanism by which folate deficiency influences carcinogenicity, we have analyzed the effect of folate deficiency on base excision repair (BER), the pathway responsible for repairing uracil in DNA. We observe an up-regulation in initiation of BER in liver of the folate-deficient mice, as evidenced by an increase in uracil DNA glycosylase protein (30%, p < 0.01) and activity (31%, p < 0.05). However, no up-regulation in either BER or its rate-determining enzyme, DNA polymerase β (β-pol) is observed in response to folate deficiency. Accordingly, an accumulation of repair intermediates in the form of DNA single strand breaks (37% increase, p < 0.03) is observed. These data indicate that folate deficiency alters the balance and coordination of BER by stimulating initiation without subsequently stimulating the completion of repair, resulting in a functional BER deficiency. In directly establishing that the inability to induce β-pol and mount a BER response when folate is deficient is causative in the accumulation of toxic repair intermediates, β-pol-haploin-sufficient mice subjected to folate deficiency displayed additional increases in DNA single strand breaks (52% increase, p < 0.05) as well as accumulation in aldehydic DNA lesions (38% increase, p < 0.01). Since young β-polhaploinsufficient mice do not spontaneously exhibit increased levels of these repair intermediates, these data demonstrate that folate deficiency and β-pol haploinsufficiency interact to increase the accumulation of DNA damage. In addition to establishing a direct role for β-pol in the phenotype expressed by folate deficiency, these data are also consistent with the concept that repair of uracil and abasic sites is more efficient than repair of oxidized bases. The mechanism by which folate deficiency influences carcinogenesis is not well established, but a phenotype of DNA strand breaks, mutations, and chromosomal instability suggests an inability to repair DNA damage. To elucidate the mechanism by which folate deficiency influences carcinogenicity, we have analyzed the effect of folate deficiency on base excision repair (BER), the pathway responsible for repairing uracil in DNA. We observe an up-regulation in initiation of BER in liver of the folate-deficient mice, as evidenced by an increase in uracil DNA glycosylase protein (30%, p < 0.01) and activity (31%, p < 0.05). However, no up-regulation in either BER or its rate-determining enzyme, DNA polymerase β (β-pol) is observed in response to folate deficiency. Accordingly, an accumulation of repair intermediates in the form of DNA single strand breaks (37% increase, p < 0.03) is observed. These data indicate that folate deficiency alters the balance and coordination of BER by stimulating initiation without subsequently stimulating the completion of repair, resulting in a functional BER deficiency. In directly establishing that the inability to induce β-pol and mount a BER response when folate is deficient is causative in the accumulation of toxic repair intermediates, β-pol-haploin-sufficient mice subjected to folate deficiency displayed additional increases in DNA single strand breaks (52% increase, p < 0.05) as well as accumulation in aldehydic DNA lesions (38% increase, p < 0.01). Since young β-polhaploinsufficient mice do not spontaneously exhibit increased levels of these repair intermediates, these data demonstrate that folate deficiency and β-pol haploinsufficiency interact to increase the accumulation of DNA damage. In addition to establishing a direct role for β-pol in the phenotype expressed by folate deficiency, these data are also consistent with the concept that repair of uracil and abasic sites is more efficient than repair of oxidized bases. In human studies, folate deficiency is associated with cancers of the lung, cervix, brain, esophagus, pancreas, breast, colon, and liver (reviewed in Refs. 1Glynn S.A. Albanes D. Nutr. Cancer. 1994; 22: 101-119Crossref PubMed Scopus (128) Google Scholar and 2Choi S.W. Mason J.B. J. Nutr. 2000; 130: 129-132Crossref PubMed Scopus (803) Google Scholar). In support of the epidemiology, the role of folate in the development of both colon and liver cancer has been experimentally demonstrated in animal studies. Folate deficiency enhances the carcinogenic effect of dimethylhydrazine (3Cravo M.L. Mason J.B. Dayal Y. Hutchinson M. Smith D. Selhub J. Rosenberg I.H. Cancer Res. 1992; 52: 5002-5006PubMed Google Scholar), whereas folate supplementation is protective (4Kim Y.I. Salomon R.N. Graeme-Cook F. Choi S.W. Smith D.E. Dallal G.E. Mason J.B. Gut. 1996; 39: 732-740Crossref PubMed Scopus (154) Google Scholar). Folate/methyl deficiency results in hepatocarcinogenesis (5Hoover K.L. Lynch P.H. Poirier L.A. J. Natl. Cancer Inst. 1984; 73: 1327-1336PubMed Google Scholar). Additionally, livers of folate/methyl-deficient rats accumulate preneoplastic changes in response to folate deficiency (6James S.J. Miller B.J. Basnakian A.G. Pogribny I.P. Pogribna M. Muskhelishvili L. Carcinogenesis. 1997; 18: 287-293Crossref PubMed Scopus (97) Google Scholar). It is suggested that the carcinogenic properties of folate deficiency are related to a reduction in S-adenosylmethionine levels altering DNA methylation status and/or to depletion of thymidylate resulting in increased uracil content of DNA. Additionally, folate is required for de novo purine biosynthesis. Which of these factors may be responsible for inducing carcinogenesis is presently unknown. Whereas the underlying mechanism connecting folate deficiency to cancer remains unknown, it is clear that folate deficiency induces a phenotype suggestive of an inability to repair DNA damage. The accumulation of strand breaks, mutations, and chromosomal instability observed in response to folate deficiency all suggest that DNA repair capacity is inhibited. In support of this, folate-deficient cells and animals inefficiently repair alkylation damage (7Branda R.F. Hacker M. Lafayette A. Nigels E. Sullivan L. Nicklas J.A. O'Neill J.P. Environ. Mol. Mutagen. 1998; 32: 33-38Crossref PubMed Scopus (16) Google Scholar). Folate deficiency acts synergistically with ethane methyl sulfonate in Chinese hamster ovary cells (8Branda R.F. Lafayette A.R. O'Neill J.P. Nicklas J.A. Mutation Res. 1999; 427: 79-87Crossref PubMed Scopus (13) Google Scholar), suggesting an inability to repair ethane methyl sulfonate-induced damage. Folate depletion in human lymphocytes sensitizes to oxidative damage induced by hydrogen peroxide (9Duthie S.J. Hawdon A. FASEB J. 1998; 12: 1491-1497Crossref PubMed Scopus (262) Google Scholar). Human colon epithelial cells grown in folate-deficient medium are unable to repair damages induced by methylmethane sulfonate and hydrogen peroxide (10Duthie S.J. Narayanan S. Blum S. Pirie L. Brand G.M. Nutr. Cancer. 2000; 37: 245-251Crossref PubMed Scopus (183) Google Scholar). Folate deficiency impairs the ability of neurons and colonocytes to repair DNA damage (11Kruman I.I. Kumaravel T.S. Lohani A. Pedersen W.A. Cutler R.G. Druman Y. Haughey N. Lee J. Evans M. Mattson M.P. J. Neurosci. 2002; 22: 1752-1762Crossref PubMed Google Scholar, 12Choi S.W. Kim Y.I. Weitzel J.N. Mason J.B. Gut. 1998; 4: 93-99Crossref Scopus (115) Google Scholar). These data suggest that the pathway responsible for repairing these damages may be ineffective when folate is limiting by demonstrating a persistence of DNA damage but stop short of directly measuring DNA repair capacity. The objectives of this study are to directly measure the effects of folate deficiency on DNA repair capacity and to begin identifying the molecular mechanisms responsible for precipitating a phenotype of cancer susceptibility when folate is deficient. Uracil has been shown to accumulate in response to folate deficiency (9Duthie S.J. Hawdon A. FASEB J. 1998; 12: 1491-1497Crossref PubMed Scopus (262) Google Scholar, 10Duthie S.J. Narayanan S. Blum S. Pirie L. Brand G.M. Nutr. Cancer. 2000; 37: 245-251Crossref PubMed Scopus (183) Google Scholar, 13Wickramasinghe S.N. Fida S. Blood. 1994; 83: 1656-1661Crossref PubMed Google Scholar, 14Blount B.C. Mack M.M. Wehr C.M. MacGregor J.T. Hiatt R.A. Wang G. Wickramasinghe S.N. Everson R.B. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3290-3295Crossref PubMed Scopus (1215) Google Scholar, 15Duthie S.J. Grant G. Narayanan S. Br. J. Cancer. 2000; 83: 1532-1537Crossref PubMed Scopus (62) Google Scholar). The DNA repair pathway for removal of uracil is the base excision repair (BER) 1The abbreviations used are: BER, base excision repair; UDG, uracil-DNA glycosylase; Ape, AP endonuclease; β-pol, polymerase β; ASB, aldehyde-reactive probe slot blot; ADL, aldehydic DNA lesion; DTT, dithiothreitol; I.D.V., integrated density value; ROPS, random oligonucleotideprimed synthesis; TEMPO, 2,2,6,6-tetramethylpiperidin-oxyl. pathway. The BER pathway repairs small, non-helix-distorting lesions in the DNA. In the process of repairing uracil, the following sequence of events occurs. Uracil-DNA glycosylase (UDG), a monofunctional glycosylase, excises uracil from the DNA backbone, creating a transient abasic site. AP endonuclease 1 (Ape1) cleaves the DNA backbone to allow for incorporation of a correct nucleotide by DNA polymerase β (β-pol). This step results in the transient formation of both a 3′-OH-containing DNA single strand break and a deoxyribose phosphate flap containing an aldehydic group. β-pol also performs the rate-limiting step of deoxyribose flap excision (16Srivastava D.K. Vande Berg B.J. Prasad R. Molina J.T. Beard W.A. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1998; 273: 21203-21209Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar), at which point only the 3′-OH group remains until ligation occurs (ligase 1 or ligase 3-XRCC1 complex), at which point the strand break is fully resolved. Typically, BER functions as a tightly coordinated sequence of enzymatic events such that damage is removed, repair is completed, and intermediate products generated during repair do not accumulate. Mice with gene-targeted disruptions in the rate-limiting step of BER exhibit a dysregulation in this coordination and accumulate DNA single strand breaks both spontaneously and in response to carcinogen exposure, have a reduced DNA damage threshold, and exhibit genomic instability in the forms of both mutation induction and chromosomal damage (17Cabelof D.C. Guo Z. Raffoul J.J. Sobol R.W. Wilson S.H. Richardson A. Heydari A.R. Cancer Res. 2003; 63: 5799-5807PubMed Google Scholar). The phenotype of folate deficiency includes accumulation of strand breaks (9Duthie S.J. Hawdon A. FASEB J. 1998; 12: 1491-1497Crossref PubMed Scopus (262) Google Scholar, 10Duthie S.J. Narayanan S. Blum S. Pirie L. Brand G.M. Nutr. Cancer. 2000; 37: 245-251Crossref PubMed Scopus (183) Google Scholar, 15Duthie S.J. Grant G. Narayanan S. Br. J. Cancer. 2000; 83: 1532-1537Crossref PubMed Scopus (62) Google Scholar, 18Melnyk S. Pogribna M. Miller B.J. Basnakian A.G. Pogribny I.P. James S.J. Cancer Lett. 1999; 146: 35-44Crossref PubMed Scopus (96) Google Scholar, 19Kim Y.I. Shirwadkar S. Choi S.W. Puchyr M. Wang Y. Mason J.B. Gastroenterology. 2000; 119: 151-161Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), reduced tolerance to DNA-damaging agents, increased mutation induction (7Branda R.F. Hacker M. Lafayette A. Nigels E. Sullivan L. Nicklas J.A. O'Neill J.P. Environ. Mol. Mutagen. 1998; 32: 33-38Crossref PubMed Scopus (16) Google Scholar, 8Branda R.F. Lafayette A.R. O'Neill J.P. Nicklas J.A. Mutation Res. 1999; 427: 79-87Crossref PubMed Scopus (13) Google Scholar, 20Branda R.F. Lafayette A.R. O'Neill J.P. Nicklas J.A. Cancer Res. 1997; 57: 2586-2588PubMed Google Scholar), and chromosomal instability (21MacGregor J.T. Schlegel R. Wehr C.M. Alperin P. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9962-9965Crossref PubMed Scopus (69) Google Scholar, 22Everson R.B. Wehr C.M. Erexson G.L. MacGregor J.T. J. Natl. Cancer Inst. 1988; 80: 525-529Crossref PubMed Scopus (109) Google Scholar) (i.e. folate deficiency induces a phenotype identical to BER deficiency). The objective of this study was to directly determine the effect of folate deficiency on base excision repair capacity. Because BER is DNA damage-inducible (23Fornace Jr., A.J. Zmudzka B. Hollander M.C. Wilson S.H. Mol. Cell. Biol. 1989; 9: 851-853Crossref PubMed Scopus (155) Google Scholar, 24Cabelof D.C. Raffoul J.J. Yanamadala S. Guo Z. Heydari A.R. Carcinogenesis. 2002; 23: 1419-1425Crossref PubMed Google Scholar), we expected to observe an induction in the activity of this repair pathway in response to the DNA damage induced by folate deficiency. However, in response to folate deficiency we fail to observe an up-regulation in either BER or the rate-determining enzyme in the pathway, β-pol. This lack of response by the BER pathway should result in an accumulation of the DNA damage induced by folate deficiency. We provide evidence that the initial step of BER, glycosylase-initiated removal of uracil, is up-regulated in response to folate deficiency, without a coordinating increase in activity of the subsequent steps of repair. This dysregulation of BER induces a state of BER deficiency mimicking that observed in mice heterozygous for β-pol. Our data demonstrate that in response to folate deficiency repair is initiated but not completed, resulting in an accumulation of DNA repair intermediate products that are genotoxic (25Horton J.K. Joyce-Gray D.F. Pachkowski B.F. Swenberg J.A. Wilson S.H. DNA Repair. 2003; 2: 27-48Crossref PubMed Scopus (83) Google Scholar). Since BER deficiency increases cancer susceptibility, this functional BER deficiency in response to folate deficiency may provide an important mechanistic explanation for the increased cancer risk associated with folate deficiency. As such, human polymorphisms and functional mutations within the β-pol gene may interact with folate deficiency to increase cancer risk. This hypothesis is supported by our data demonstrating that folate deficiency results in a greater accumulation of DNA damage in mice haploinsufficient for β-pol. Experiments were performed in young (3–4 months) male C57BL/6-specific pathogen-free mice in accordance with the National Institutes of Health guidelines for the use and care of laboratory animals. The Wayne State University Animal Investigation Committee approved the animal protocol. Mice were maintained on a 12-h light/dark cycle and fed standard mouse chow and water ad libitum. Mice heterozygous for the DNA polymerase β gene (β-pol+/–) were created in Rajewsky's laboratory by deletion of the promoter and the first exon of the β-pol gene (26Gu H. Marth J.D. Orban P.C. Mossmann H. Rajewsky K. Science. 1994; 265: 103-106Crossref PubMed Scopus (1182) Google Scholar). 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 as described previously (17Cabelof D.C. Guo Z. Raffoul J.J. Sobol R.W. Wilson S.H. Richardson A. Heydari A.R. Cancer Res. 2003; 63: 5799-5807PubMed Google Scholar). At 3–4 months of age, 20 β-pol+/+ and 20 β-pol+/– mice were randomly assigned to two dietary groups and were fed AIN93G-purified isoenergetic diets. (Dyets, Inc., Lehigh Valley, PA). The control group received a folate adequate diet containing 2 mg/kg folic acid. The experimental group received a folate-deficient diet containing 0 mg/kg folic acid. Diets were stored at –20 °C. 1% succinyl sulfathiazole was added to all diets. The animals' food intake and body weights were monitored twice weekly to monitor for signs of toxicity (i.e. weight loss), and the experimental diets were continued for 8 weeks. Animals were anesthetized under CO2 and sacrificed by cervical dislocation. Whole blood was collected, and tissues were flash frozen and stored in liquid nitrogen. Serum folate levels were measured using the SimulTRAC-SNB radioassay kit for vitamin B12 (57Co) and folate (125I) per the manufacturer's protocol (ICN Diagnostics, Orangeburg, NY). Blood was collected at time of sacrifice and allowed to clot at room temperature for 60 min. Samples were centrifuged, and serum was collected for immediate analysis of serum folate levels. Standards provided in the kit were used to generate a standard curve for determination of sample folate values. Radioactivity was measured by a γ-counter, and values were calculated as described by the manufacturer for both serum folate and serum B12. TEMPO Extraction of DNA—Liver DNA for the aldehyde-reactive probe slot blot (ASB) assay was extracted according to the method described by Hofer and Moller (27Hofer T. Moller L. Chem. Res. Toxicol. 1998; 11: 882-887Crossref PubMed Scopus (75) Google Scholar) with some modifications. This method minimizes artifactual DNA damage by using 20 mm TEMPO in all solutions and reagents and by minimizing heat treatment of DNA. Briefly, 100 mg of liver tissue was homogenized in 5 ml of ice-cold phosphate-buffered saline with 20 mm TEMPO and centrifuged at 2000 × g at 4 °C for 5 min. Supernatant was decanted, and pellet was resuspended in 2.5 ml of lysis buffer (pH 8.0; Applied Biosystems, Foster City, CA) with 20 mm TEMPO. Proteinase K (30 units; Ambion, Austin, TX) was added, and samples were incubated overnight at 4 °C. DNA was extracted with 2.5 ml 70% phenol/water/chloroform (Applied Biosystems, Foster City, CA) with 20 mm TEMPO. Further extraction with 2.5 ml of sevag (chloroform/isoamyl alcohol, 24:1) with 20 mm TEMPO was completed. DNA was precipitated using 7.5% 4 m NaCl and 2 volumes of 100% cold ethanol, and pellet was washed in 70% ethanol. The pellet was resuspended in 700 μl of phosphate-buffered saline with 20 mm TEMPO and rehydrated at 4 °C. The samples were then treated with RNase A (2 μg) and RNase T1 (1,000 units; Ambion, Austin, TX) at 37 °C for 30 min to digest the RNA contamination. DNA was cold ethanol-precipitated and resuspended in 400 μl of deionized water at 4 °C. DNA was stored at –70 °C. Gravity Tip Column Extraction of DNA—DNA for the random oligonucleotide-primed synthesis (ROPS) assay was isolated using Qiagen (Valencia, CA) gravity tip columns as described in the manufacturer's protocol. This method generates large fragments of DNA (up to 150 kb) while minimizing shearing. Detection of aldehydic DNA lesions (ADLs) was carried out by ASB as described previously (28Nakamura J. Walker V.E. Upton P.B. Chiang S.Y. Kow Y.W. Swenberg J.A. Cancer Res. 1998; 58: 222-225PubMed Google Scholar) with slight modifications. DNA (8 μg) was incubated in 30 μl of phosphate-buffered saline with 2 mm aldehyde reactive probe (Dojindo Laboratories, Kumamoto, Japan) at 37 °C for 10 min. DNA was precipitated by the cold ethanol method (described above) and resuspended in 1× TE buffer overnight at 4 °C. DNA was heat-denatured at 100 °C for 10 min, quickly chilled on ice, and mixed with an equal volume of 2 m ammonium acetate. The nitrocellulose membrane (Schleicher & Schuell) was prewet in deionized water and washed for 10 min in 1 mm ammonium acetate. DNA was immobilized on the pretreated nitrocellulose membrane using an Invitrogen filtration manifold system. The membrane was washed in 5× SSC for 15 min at 37 °C and then baked under vacuum at 80 °C for 30 min. The dried membrane was incubated in a hybridization buffer (20 mm Tris, pH 7.5, 0.1 m NaCl, 1 mm EDTA, 0.5% (w/v) casein, 0.25% (w/v) bovine serum albumin, 0.1% (v/v) Tween 20) for 30 min at room temperature. The membrane was then incubated in fresh hybridization buffer containing 100 μl of streptavidin-conjugated horseradish peroxidase (BioGenex, San Ramon, CA) at room temperature for 45 min. Following incubation in horseradish peroxidase, the membrane was washed three times for 5 min each at 37 °C in TBS, pH 7.5 (0.26 m NaCl, 1 mm EDTA, 20 mm Tris, pH 7.5, 0.1% Tween 20). Membrane was incubated in ECL (Pierce) for 5 min at room temperature and visualized using a ChemiImager™ system (AlphaInnotech, San Leandro, CA). Nuclear proteins for Western analyses and enzymatic activity assays were isolated using the Sigma CelLytic™ NuCLEAR™ extraction kit, a method that disrupts cells with hypotonic buffer, allowing the cytoplasmic fraction to be removed while the nuclear proteins are released from the nuclei by a high salt buffer. All samples and tubes were handled and chilled on ice, and all solutions were made fresh according to the manufacturer's protocol. The extract was snap frozen in liquid nitrogen and stored at –70 °C. In order to remove salt, the crude nuclear extract was dialyzed against 1 liter of dialysis buffer (20 mm Tris-HCl, pH 8.0, 100 mm KCl, 10 mm NaS2O5, 0.1 mm DTT, 0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A) for 4–6 h at 4 °C using Slide-A-Lyzer® mini-dialysis units suspended in a floatation device (Pierce). The dialyzed nuclear extracts were flash frozen in liquid nitrogen and stored at –70 °C. Protein concentrations of the nuclear extracts were determined according to Bradford using Protein Assay Kit I (Bio-Rad). Western analysis was performed using liver nuclear extracts (50 μg) subjected to 10% SDS-PAGE and transferred to a Hybond™ ECL™ nitrocellulose membrane (Amersham Biosciences) using a Bio-Rad semidry transfer apparatus. Prior to hybridization, the membranes were stained with MemCode (Pierce) to ensure equal transfer of protein to the membrane. Western blot analysis was accomplished using manufacturer-recommended dilutions of antisera developed against UDG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), p53 (polyclonal antibody 240; Santa Cruz Biotechnology), β-pol (Ab-1 Clone 18S; NeoMarkers, Fremont, CA), and Ape/Ref1 (Novus Biologicals, Littleton, CO). The bands were detected and quantified using a ChemiImager™ system (AlphaInnotech) after incubation in SuperSignal® West Pico chemiluminescent substrate (Pierce). The data are expressed as the integrated density value (I.D.V.) of the band per μg of protein loaded. UDG activity was determined as described by Stuart et al. (29Stuart J.A. Karahalil B. Hogue B.A. Souza-Pinto N.C. Bohr V.A. FASEB J. 2004; 18: 595-597Crossref PubMed Scopus (97) Google Scholar). Briefly, the 20-μl reaction contained 70 mm Hepes (pH 7.5), 1 mm EDTA, 1 mm DTT, 75 mm NaCl, 0.5% bovine serum albumin, 90 fmol of 5′-end-labeled single-stranded uracil-containing oligonucleotide that was 3′-protected by an amino-spacer (5′-ATATACCGCGGUCGGCCGATCAAGCTTATT-3′, MIDLAND, Midland, TX), and 5 μg of liver nuclear extract. Reactions were incubated at 37 °C for 1 h and then terminated by the addition of 5 μg of proteinase K and 1 μl of 10% SDS and incubation at 55 °C for 30 min. DNA was precipitated in glycogen, ammonium acetate, and ethanol at –20 °C overnight, resuspended in a loading buffer containing 80% formamide, 10 mm EDTA, and 1 μg/ml each of bromphenol blue and xylene cyanol FF. Substrate and reaction products were separated on a 20% denaturing sequencing gel. Glycosylase activity (presence of an 11-mer band) was visualized and quantified using a Molecular Imager® System (Bio-Rad) by calculating the relative amount of the 11-mer oligonucleotide product with the unreacted 30-mer substrate (product/product + substrate). The data are expressed as machine counts/μg of protein. Negative controls consisted of the reaction mixture and oligonucleotide in the absence of nuclear extract. 1 unit of uracil DNA glycosylase inhibitor was added to one sample in each reaction to demonstrate that incision activity was the result of UDG specifically and not another uracil-specific glycosylase (i.e. SMUG). The 5′-endonuclease activity of Ape was analyzed using a quantitative in vitro assay that measures the incision of a 26-mer duplex oligonucleotide substrate containing a tetrahydrofuran (F) AP site as previously described (30Wilson III, D.M. Takeshita M. Grollman A.P. Demple B. J. Biol. Chem. 1995; 270: 16002-16007Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). A 26-mer oligonucleotide (5′-AATTCACCGGTACCFTCTAGAATTCG-3′) was 5′-end-labeled and annealed to an equimolar amount of the complementary strand (5′-CGAATTCTAGAGGGTACCGGTGAATT-3′). 2.5 pmol of double strand oligonucleotide was incubated with 100 ng of crude nuclear extract from liver of control and folate-deficient mice for 15 min at 37 °C and stopped by the addition of 50 mm EDTA (reaction mixture; final concentrations: 50 mm Hepes, pH 7.5, 50 mm KCl, 10 mm MgCl2, 2 mm DTT, 1 μg/ml bovine serum albumin, and 0.05% Triton X-100). Reaction products were run on a 15% denaturing sequencing gel. Endonuclease activity (presence of a 14-mer band) was visualized and quantified using a Molecular Imager® System (Bio-Rad) by calculating the relative amount of the 14-mer oligonucleotide product with the unreacted 26-mer substrate (product/product + substrate). The data are expressed as machine counts/ng of protein. BER capacity was determined as described previously (17Cabelof D.C. Guo Z. Raffoul J.J. Sobol R.W. Wilson S.H. Richardson A. Heydari A.R. Cancer Res. 2003; 63: 5799-5807PubMed Google Scholar). Briefly, end-labeled and purified 30-bp oligonucleotides (upper strand, 5′-ATATACCGCGGUCGGCCGATCAAGCTTATT-3′; lower strand, 3′-TATATGGCGCCGGCCGGCTAGTTCGAATAA-5′) containing a G:U mismatch and an HpaII restriction site (GGCC) and protected by a 3′ amino spacer were incubated in a reaction mixture (100 mm Tris-HCl, pH 7.5, 5mm MgCl2,1mm DTT, 0.1 mm EDTA, 2 mm ATP, 0.5 mm NAD, 20 μm dNTPs, 5 mm di-Tris-phosphocreatine, 10 units of creatine phosphokinase) with 50 μg of nuclear extract isolated from liver of control and folate-deficient mice. The reaction mixtures were incubated for 30 min at 37 °C, followed by 5 min at 95 °C to stop the reaction. The duplex oligonucleotides were allowed to reanneal for 1 h at room temperature and spun down to pellet the denatured proteins. The duplex oligonucleotides present in the supernatant were treated with 20 units of HpaII (Promega, Madison, WI) for 1 h at 37 °C and separated by electrophoresis on a 20% denaturing sequencing gel. Repair activity (presence of a 16-mer band) is visualized and quantified using a Molecular Imager® System (Bio-Rad) by calculating the ratio of the 16-mer oligonucleotide product with the 30-mer substrate (product/substrate). The data are expressed as machine counts/μg of protein. The relative number of 3′-OH group-containing DNA strand breaks was quantified using a Klenow(exo–) incorporation assay based on the ability of Klenow to initiate DNA synthesis from a 3′-OH (31Basnakian A.G. James S.J. DNA Cell Biol. 1996; 15: 255-262Crossref PubMed Scopus (50) Google Scholar). DNA was heat-denatured at 100 °C for 5 min, and 0.25 μg of DNA was added to 15 μl of a Klenow reaction buffer (0.5 mm dTTP, 0.5 mm dGTP, 0.5 mm dATP, 0.33 μm dCTP, 5 units of Klenow(exo–) (New England Biolabs, Beverly, MA)) with 10× Klenow buffer per the manufacturer's protocol (New England Biolabs) and 5 μCi of [α-32P]dCTP (3000 Ci/mmol; PerkinElmer Life Sciences). Reaction mixtures were incubated at 16 °C for 30 min, and the reaction was stopped with the addition of 25 μl of 12.5 mm EDTA, pH 8.0. Samples were spotted (5 μl) onto scored and numbered Whatman DE81 chromatography paper and allowed to airdry. The chromatography paper was then washed five times for 5 min each time in 0.5 m Na2HPO4 (dibasic) to remove unincorporated [α-32P]dCTP and then rinsed twice briefly in water and allowed to air-dry. Paper was cut and placed into scintillation vials with 2.5 ml of Scinti Verse mixture (Fisher). Incorporation of [α-32P]dCTP was quantified using a Packard scintillation counter. Statistical significance between means was determined using analysis of variance followed by Fisher's least significant difference test where appropriate (32Sokal R.R. Rohlf F.J. Biometry. W.H. Freeman and Co., New York1981: 169-176Google Scholar). A p value less than 0.05 was considered statistically significant. In an extensive study, we have carefully characterized the effect of folate deficiency on weight gain/loss and plasma folate levels in C57BL/6 mice. Throughout the 8-week feeding study, the animals' food intake and body weights were monitored twice weekly. Long term folate deficiency can result in toxicity, evidenced primarily by weight loss. Importantly, in our studies, no difference in food intake or weight gain/loss was observed (Fig. 1A). It is essential to demonstrate that the experimental diet (0 mg/kg folic acid) resulted in decreased serum folate levels. As expected, a significant decrease in the level of serum folate in the folate-deficient mouse was observed. The 0 mg/kg folic acid group had serum folate levels 93% lower than the control animals (p < 0.001), such that the folate levels of the deficient animals approached zero (Fig. 1B). The addition of 1% succinyl sulfathiazole allowed attainment of this severity of folate deficiency by preventing intestinal production of folates. Since vitamin B12 can alter one-carbon metabolism through its participation in the methionine synthase reaction, it was important to determine whether our results might be confounded by changes in serum B12 levels. Importantly, no differences were observed in B12 levels between control and deficient groups (data not shown). There was no effect of β-pol heterozygosity on weight or serum folate levels in response to folate deficiency (data not shown). This is the first investigation to directly measure the effect of folate deficiency on BER capacity. It is the BER pathway that holds primary responsibility for removing folate-induced damage from DNA. Previously we (24Cabelof D.
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