Protection against Methylation-induced Cytotoxicity by DNA Polymerase β-Dependent Long Patch Base Excision Repair
2000; Elsevier BV; Volume: 275; Issue: 3 Linguagem: Inglês
10.1074/jbc.275.3.2211
ISSN1083-351X
AutoresJulie K. Horton, Rajendra Prasad, Esther W. Hou, Samuel H. Wilson,
Tópico(s)Epigenetics and DNA Methylation
ResumoUsing a plasmid-based uracil-containing DNA substrate, we found that the long patch base excision repair (BER) activity of a wild-type mouse fibroblast extract was partially inhibited by an antibody to DNA polymerase β (β-pol). This suggests that β-pol participates in long patch BER, in addition to single-nucleotide BER. In single-nucleotide BER, the deoxyribose phosphate (dRP) in the abasic site is removed by the lyase activity of β-pol. Methoxyamine (MX) can react with the aldehyde of an abasic site, making it refractory to the β-elimination step of the dRP lyase mechanism, thus blocking single-nucleotide BER. MX exposure sensitizes wild-type, but not β-pol null mouse embryonic fibroblasts, to the cytotoxic effects of methyl methanesulfonate (MMS) and methylnitrosourea. Expression of β-pol in the null cells restores the ability of MX to modulate sensitivity to MMS. The β-pol null cells are known to be hypersensitive to MMS and methylnitrosourea, and in the presence of MX (i.e. under conditions where single-nucleotide BER is blocked) the null cells are still considerably more sensitive than wild-type. The data are consistent with a role of β-pol in long patch BER, which helps protect cells against methylation damage-induced cytotoxicity. Using a plasmid-based uracil-containing DNA substrate, we found that the long patch base excision repair (BER) activity of a wild-type mouse fibroblast extract was partially inhibited by an antibody to DNA polymerase β (β-pol). This suggests that β-pol participates in long patch BER, in addition to single-nucleotide BER. In single-nucleotide BER, the deoxyribose phosphate (dRP) in the abasic site is removed by the lyase activity of β-pol. Methoxyamine (MX) can react with the aldehyde of an abasic site, making it refractory to the β-elimination step of the dRP lyase mechanism, thus blocking single-nucleotide BER. MX exposure sensitizes wild-type, but not β-pol null mouse embryonic fibroblasts, to the cytotoxic effects of methyl methanesulfonate (MMS) and methylnitrosourea. Expression of β-pol in the null cells restores the ability of MX to modulate sensitivity to MMS. The β-pol null cells are known to be hypersensitive to MMS and methylnitrosourea, and in the presence of MX (i.e. under conditions where single-nucleotide BER is blocked) the null cells are still considerably more sensitive than wild-type. The data are consistent with a role of β-pol in long patch BER, which helps protect cells against methylation damage-induced cytotoxicity. base excision repair DNA polymerase β flap endonuclease 1 5′-deoxyribose phosphate apurinic/apyrimidinic AP endonuclease uracil-DNA glycosylase methoxyamine methyl methanesulfonate methylnitrosourea dithiothreitol nucleotide excision repair Single base lesions in mammalian genomic DNA are repaired by the process known as base excision repair (BER).1 Typically, such base lesions arise through endogenous events including spontaneous base loss, uracil incorporation, base deamination, and base oxidation, and also through base alkylations introduced by alkylating agents. Recent evidence has indicated that BER in mammalian cells is mediated through at least two subpathways that are differentiated by the repair patch sizes and the enzymes involved and are designated as “single-nucleotide BER” and “long patch BER” (2–10- nucleotide patch) (1.Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (265) Google Scholar, 2.Frosina G. Fortini P. Rossi O. Carrozzino F. Raspaglio G. Cox L.S. Lane D.P. Abbondandolo A. Dogliotti E. J. Biol. Chem. 1996; 271: 9573-9578Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar, 3.Wilson S.H. Mutat. Res. 1998; 407: 203-215Crossref PubMed Scopus (265) Google Scholar). The most simple single-nucleotide BER pathway is an ordered sequential process initiated by base excision by a monofunctional glycosylase. Removal of the deoxyribose phosphate (dRP) in the abasic site is by the dRP lyase activity of DNA polymerase β (β-pol) (4.Srivastava 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). Single-nucleotide BER of a uracil-containing oligonucleotide substrate can be reconstituted in vitro with four purified human proteins: uracil-DNA glycosylase (UDG), apurinic/apyrimidinic endonuclease (APE), β-pol, and DNA ligase I (4.Srivastava 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), and a complex containing these enzymes can be isolated from extracts of mammalian cells (5.Prasad R. Singhal R.K. Srivastava D.K. Molina J.T. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1996; 271: 16000-16007Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). In addition, genetic evidence with β-pol- and DNA ligase I-deficient mammalian cell lines indicates that these proteins are important for the repair of alkylating agent induced lesions (6.Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (794) Google Scholar, 7.Teo I.A. Arlett C.F. Harcourt S.A. Priestley A. Broughton B.C. Mutat. Res. 1983; 107: 371-386Crossref PubMed Scopus (53) Google Scholar). The choice of subpathway in BER (single-nucleotide or long patch) depends on whether the dRP intermediate can be efficiently removed by the β-pol lyase activity to yield a 5′-phosphorylated DNA strand capable of serving as a substrate for DNA ligase (4.Srivastava 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, 8.Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (268) Google Scholar). When such processing is not efficient, for example during repair of a reduced abasic site, long patch BER can occur (9.Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (678) Google Scholar). In addition, it has been suggested that the properties of the damage-specific DNA glycosylase (e.g., monofunctional, exhibiting only glycosylase activity, or bifunctional, having glycosylase and β-lyase activity) can determine the BER subpathway (10.Fortini P. Parlanti E. Sidorkina O.M. Laval J. Dogliotti E. J. Biol. Chem. 1999; 274: 15230-15236Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). For BER studied in vitro, the two subpathways can co-repair the same type of DNA lesion and operate in the same extract (10.Fortini P. Parlanti E. Sidorkina O.M. Laval J. Dogliotti E. J. Biol. Chem. 1999; 274: 15230-15236Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 11.Fortini P. Pascucci B. Parlanti E. Sobol R.W. Wilson S.H. Dogliotti E. Biochemistry. 1998; 37: 3575-3580Crossref PubMed Scopus (200) Google Scholar). It is still unclear which enzymes are involved in vivo in the various BER subpathways. Biochemical and genetic evidence indicate that, in addition to those mentioned above, 3-methyladenine DNA glycosylase, XRCC1, DNA ligase III, flap endonuclease 1 (FEN1), and poly(ADP-ribose) polymerase are important proteins for BER and the repair of alkylating agent-induced lesions (9.Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (678) Google Scholar, 12.Engelward B.P. Dreslin A. Christensen J. Huszar D. Kurahara C. Samson L. EMBO J. 1996; 15: 945-952Crossref PubMed Scopus (184) Google Scholar, 13.Cappelli E. Taylor R. Cevasco M. Abbondandolo A. Caldecott K. Frosina G. J. Biol. Chem. 1997; 272: 23970-23975Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 14.Tebbs R.S. Flannery M.L. Meneses J.J. Hartmann A. Tucker J.D. Thompson L.H. Cleaver J.E. Pedersen R.A. Dev. Biol. 1999; 208: 513-529Crossref PubMed Scopus (304) Google Scholar, 15.Kim K. Biade S. Matsumoto Y. J. Biol. Chem. 1998; 273: 8842-8848Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 16.Satoh M.S. Lindahl T. Nature. 1992; 356: 356-358Crossref PubMed Scopus (988) Google Scholar, 17.Trucco C. Oliver F.J. de Murcia G. Menissier-de Murcia J. Nucleic Acids Res. 1998; 26: 2644-2649Crossref PubMed Scopus (314) Google Scholar). For single-nucleotide repair, β-pol is the polymerase of choice for the resynthesis step (6.Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (794) Google Scholar); however, β-pol-deficient extracts are able to perform single-nucleotide BER (11.Fortini P. Pascucci B. Parlanti E. Sobol R.W. Wilson S.H. Dogliotti E. Biochemistry. 1998; 37: 3575-3580Crossref PubMed Scopus (200) Google Scholar). For long patch BER, repair has been shown to be stimulated by proliferating cell nuclear antigen which has led to the suggestion that DNA polymerases δ and/or ε are involved (1.Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (265) Google Scholar, 18.Stucki M. Pascucci B. Parlanti E. Fortini P. Wilson S.H. Hubscher U. Dogliotti E. Oncogene. 1998; 17: 835-843Crossref PubMed Scopus (162) Google Scholar). However, the role of proliferating cell nuclear antigen may be limited to stimulation of FEN1-dependent flap cleavage (19.Gary R. Kim K. Cornelius H.L. Park M.S. Matsumoto Y. J. Biol. Chem. 1999; 274: 4354-4363Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), and proliferating cell nuclear antigen can promote β-pol-dependent long patch repair by this mechanism (9.Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (678) Google Scholar). There is additional evidence pointing to a role of β-pol in long patch repair DNA synthesis. For example, β-pol antibody was found to inhibit long patch repair mediated by cell extracts (9.Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (678) Google Scholar), and β-pol-deficient extracts were unable to repair a reduced AP site on a linear DNA substrate that could be repaired by wild-type extracts (20.Biade S. Sobol R.W. Wilson S.H. Matsumoto Y. J. Biol. Chem. 1998; 273: 898-902Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Additionally, recent data indicate that β-pol plays an essential role in the strand displacement synthesis of long patch BER (21.Dianov G.L. Prasad R. Wilson S.H. Bohr V.A. J. Biol. Chem. 1999; 274: 13741-13743Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 22.Prasad R. Dianov G.L. Bohr V.A. Wilson S.H. J. Biol. Chem. 2000; 275 (in press)Abstract Full Text Full Text PDF Scopus (191) Google Scholar). In this paper, we describe further analysis of the role of β-pol in BER and dissect the contribution of β-pol in protecting cells against the cytotoxic effects of simple methylating agents. Using wild-type mouse embryonic fibroblast cell extracts, we find that repair of a plasmid-based uracil-containing BER substrate by long patch BER is partially blocked by an antibody to β-pol. Thus, this extract is capable of both β-pol-dependent and β-pol-independent long patch BER, in addition to the single-nucleotide BER we have described previously (6.Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (794) Google Scholar). We also examined the cellular phenotype of a β-pol-dependent long patch BER deficiency in mouse fibroblast cells. First, single-nucleotide BER was chemically blocked by methoxyamine (MX), and then the effect of β-pol gene deletion on cellular sensitivity to DNA-alkylating agents was examined. The results demonstrate a role of β-pol-dependent long patch BER in protection of cells against the cytoxicity of the monofunctional DNA-alkylating agents methyl methanesulfonate (MMS) and methylnitrosourea (MNU). [α-32P]dCTP, [α-32P]dATP, [α-32P]ddATP (3000 Ci/mmol), and MicroSpin S-400 HR columns were from Amersham Pharmacia Biotech. Maxi-plasmid and polymerase chain reaction purification kits were from Qiagen (Valencia, CA). NENSORB-20 columns were from NEN Life Science Products. Plasmid DNA, pUC19, was prepared from cells grown in Luria broth containing 100 μg/ml ampicillin by the procedure specified in the Qiagen plasmid kit. High performance liquid chromatography-purified synthetic oligodeoxyribonucleotides were obtained from Oligos Etc., Inc. (Wilsonville, OR) or Operon Technologies, Inc. (Alameda, CA). Phenol/chloroform, T4 DNA ligase, restriction enzymes (PstI, BamHI,BglII, HaeIII, SphI, andNcoI) and Dulbecco's modified Eagle's medium, GlutaMAX-1, and minimal essential medium non-essential amino acid solution were from Life Technologies, Inc. T4 polynucleotide kinase and terminal deoxynucleotidyltransferase were from Promega (Madison, WI). β-Nicotinamide adenine dinucleotide, creatine phosphokinase, diTris-phosphocreatine, MMS, MNU, and MX were from Sigma-Aldrich. Fetal bovine serum was obtained from Summit Biotechnology (Ft. Collins, CO) and hygromycin from Roche Molecular Biochemicals. Recombinant human β-pol, FEN1, human APE, and UDG with 84 amino acids deleted from the amino terminus were purified as described (23.Beard W.A. Wilson S.H. Methods Enzymol. 1995; 262: 98-107Crossref PubMed Scopus (161) Google Scholar, 24.Strauss P.R. Beard W.A. Patterson T.A. Wilson S.H. J. Biol. Chem. 1997; 272: 1302-1307Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 25.Slupphaug G. Eftedal I. Kavli B. Bharati S. Helle N.M. Haug T. Levine D.W. Krokan H.E. Biochemistry. 1995; 34: 128-138Crossref PubMed Scopus (251) Google Scholar). Antiserum specific for β-pol was raised by immunization of rabbits (26.Singhal R.K. Prasad R. Wilson S.H. J. Biol. Chem. 1995; 270: 949-957Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). The cell line utilized for preparation of cell extracts was a clone of the wild-type mouse embryonic fibroblast cell line Mβ16tsA described previously (6.Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (794) Google Scholar). The wild-type mouse fibroblasts, a clone of the isogenic β-pol null line Mβ19tsA (6.Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (794) Google Scholar), and the wild-type β-pol minigene-transfected null cell line, 19/A5 (27.Ochs K. Sobol R.W. Wilson S.H. Kaina B. Cancer Res. 1999; 59: 1544-1551PubMed Google Scholar), were routinely grown at 34 °C in a 10% CO2incubator in Dulbecco's modified Eagle's medium supplemented with GlutaMAX-1, 10% fetal bovine serum, and hygromycin (80 μg/ml). Primary Xpa+/− and Xpa−/− isogenic mouse embryonic fibroblasts (28.de Vries A. van Steeg H. Semin. Cancer Biol. 1996; 7: 229-240Crossref PubMed Scopus (25) Google Scholar) were obtained from Dr. Harry van Steeg (National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands). These cells were transformed with SV40 T antigen as described for the wild-type and β-pol null fibroblasts (6.Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (794) Google Scholar). Transformed Xpa fibroblasts were routinely grown at 34 °C in a 10% CO2 incubator in Dulbecco's modified Eagle's medium supplemented with glutamine, 10% fetal bovine serum, minimal essential medium non-essential amino acids, and hygromycin (80 μg/ml). All cells were routinely tested and found to be free of mycoplasma contamination. For the long patch BER assay, wild-type mouse fibroblast cells were cultured in 150-mm dishes until near-confluent, washed three times with Dulbecco's phosphate-buffered saline (Life Technologies, Inc.), and harvested by scraping. Approximately 5 × 107 cells were suspended in 1 ml of buffer (5 mm NaPO4, pH 7.1, 150 mm NaCl, 2.5 mm KCl) plus protease inhibitors, aprotinin (10 μg/ml), leupeptin (0.5 μg/ml), and Pefabloc SC (10 μg/ml) supplied by Roche Molecular Biochemicals. The suspension was subjected to five cycles of freeze/thaw and further disrupted by sequential passage through 18- and 22-gauge needles. The resulting homogenate was centrifuged at 20,000 × g for 10 min at 4 °C. The supernatant fraction was removed, aliquoted, and stored at −80 °C. Protein concentration was measured using the Bio-Rad protein assay dye reagent. For the AP endonuclease assay, cell extracts were prepared from wild-type mouse fibroblasts as described previously (20.Biade S. Sobol R.W. Wilson S.H. Matsumoto Y. J. Biol. Chem. 1998; 273: 898-902Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). A partially duplex oligonucleotide was formed by annealing a phosphorylated 12-mer (5′-ACCGGTACUGGC-3′) containing a uracil residue at position 9 and a 20-mer (5′-ACGTGCCGGTACCGGTCTAG-3′). The 12-mer (6 nmol) and 20-mer (6 nmol) were mixed in 30 μl of buffer containing 10 mmTris-HCl, pH 8.0, 1 mm EDTA, 250 mm NaCl, heated to 98 °C for 3 min, and then slowly cooled to room temperature. The annealed oligonucleotide duplex was purified by precipitation, washed twice with 70% ethanol, and resuspended in 30 μl of 10 mm Tris-HCl, pH 8.0, 1 mm EDTA. An aliquot of this DNA was analyzed by 20% non-denaturing polyacrylamide gel electrophoresis to confirm that annealing had occurred. The closed circular DNA substrate containing a unique G:U base pair was constructed according to a previously described procedure (29.Sandigursky M. Freyer G.A. Franklin W.A. Nucleic Acids Res. 1998; 26: 1282-1287Crossref PubMed Scopus (18) Google Scholar) with slight modifications. Briefly, pUC19 plasmid (500 μg) was digested with PstI (750 units) overnight at 37 °C, and the reaction was checked for complete linearization by 1% agarose gel electrophoresis. The reaction mixture was diluted with five volumes of Qiagen binding buffer, and the plasmid was purified using a Qiagen-tip 500. The purified PstI-linearized pUC19 plasmid DNA (300 pmol) was ligated to the G:U base pair-containing oligonucleotide duplex (800 pmol) in a 2-ml reaction mixture containing 100 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 100 mm NaCl, 1 mm dithiothreitol (DTT), 2 mm ATP, 800 units of PstI, and 250 units of T4 DNA ligase at 15 °C overnight. The ligation product was purified using a Qiagen-tip 500 as described above and 5′-phosphorylated in a reaction mixture (500 μl) containing 10 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 100 mm NaCl, 1 mm 2-mercaptoethanol, 2.5 mm ATP, 600 units ofBamHI, and 250 units of T4 polynucleotide kinase for 3 h at 37 °C. After purification by Qiagen-tip 500, the DNA was circularized at 15 °C overnight in a 5-ml reaction mixture containing 50 mm Tris-HCl, pH 7.5, 10 mmMgCl2, 100 mm NaCl, 1 mm DTT, 1 mm ATP, 750 units of BamHI, 750 units ofBglII, and 300 units of T4 DNA ligase. The reaction product was precipitated with three volumes of ethanol, washed twice with 70% ethanol, and resuspended in 500 μl of H2O. The circular DNA substrate was separated from linear DNA by 0.8% agarose gel electrophoresis. A small portion (1 μl) of the sample was electrophoresed as a marker in a separate lane. This marker, but not the preparative sample, was visualized by UV light, to minimize UV/ethidium bromide-induced nicking of the DNA substrate. The closed circular DNA substrate was isolated by electroelution and purified using a Qiagen polymerase chain reaction purification kit. The product was eluted in 10 mm Tris-HCl, pH 7.5, and DNA was quantified using Hoechst 33258 dye. BER assays were performed in a reaction mixture (11 μl) that contained 5 μg of cell extract, 1 nm closed circular DNA substrate, 50 mm Hepes, pH 7.5, 5 mm MgCl2, 1 mm DTT, 0.1 mm EDTA, 5 mm diTris-phosphocreatine, 10 units of creatine phosphokinase, 4 mm ATP, 0.5 mmNAD, 4 μm [α-32P]dCTP (specific activity 1 × 106 DPM/pmol), and 4 μm each dATP, TTP, and dGTP. Alternatively, 4 μm[α-32P]dATP (specific activity 1 × 106 DPM/pmol) was included as the labeled nucleoside triphosphate with 4 μm each of the remaining three unlabeled nucleoside triphosphates. After incubation at 37 °C for 60 min, the reactions were stopped by adding 1.5 μl of 0.5 mEDTA. The samples were extracted with phenol/chloroform, and the DNA was purified using a MicroSpin S-400 HR column and ethanol precipitation. The DNA samples were resuspended in 8 μl of H2O, and repair was assessed by digestion with eitherHaeIII alone or with SphI + NcoI (10 units each) at 37 °C overnight. Samples were then electrophoresed on a 20% denaturing polyacrylamide gel. Radiolabeled products were quantified using a Molecular Dynamics PhosphorImager 450 and ImageQuant software. A 49-mer oligodeoxyribonucleotide containing a uracil residue at position 21 (5.Prasad R. Singhal R.K. Srivastava D.K. Molina J.T. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1996; 271: 16000-16007Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) was labeled at the 3′ end by terminal deoxynucleotidyltransferase using [α-32P]ddATP and annealed to its complementary strand by heating the solution at 90 °C for 3 min, followed by slow cooling to 25 °C. 32P-Labeled duplex oligodeoxynucleotide was separated from unincorporated [α-32P]ddATP using a NENSORB-20 column according to the manufacturer's suggested protocol. The radiolabeled oligodeoxynucleotide was lyophilized, resuspended in H2O, and stored at −30 °C. 32P-Labeled uracil containing duplex DNA (62.5 nm) was pretreated with 10 nm UDG for 20 min at 37 °C in 100 μl of buffer containing 70 mm Hepes, pH 7.4, 0.5 mm EDTA, and 0.2 mm DTT. The reaction mixture was then supplemented with 10 mm MgCl2and APE, either 5 nm (for normal AP-DNA) or 100 nm (for MX-adducted DNA) and the incubation continued for an additional 20 min at 37 °C. To prepare MX-adducted substrate, the32P-labeled UDG-treated duplex oligonucleotide was mixed with 33 mm MX in buffer containing 50 mmKPO4, pH 7.1 and incubated at 37 °C. After 30 min, the DNA was recovered by ethanol precipitation, lyophilized, resuspended in water, and stored at −30 °C. AP endonuclease activity was assayed in a reaction mixture (10 μl) containing 50 mm Hepes, pH 7.4, 10 mm MgCl2, 2 mm DTT, and 20 nm32P-labeled MX-adducted DNA. The reaction was initiated by adding the indicated concentrations of purified APE or cell extract, and incubation was at 37 °C. Samples were withdrawn at the indicated time periods. The reaction was terminated by transfer to 0–1 °C, and the DNA product was stabilized by addition of NaBH4 to a final concentration of 340 mm and incubating 30 min on ice. The stabilized DNA products were recovered by ethanol precipitation in the presence of 0.1 μg/ml tRNA and resuspended in 10 μl of gel loading buffer (95% formamide, 20 mm EDTA, 0.02% bromphenol blue, and 0.02% xylene cyanol). Alternatively, the reactions were terminated without product stabilization by the addition of an equal volume of gel loading buffer. After incubation at 75 °C for 2 min, the reaction products were separated by electrophoresis in a 20% polyacrylamide gel containing 8m urea in 89 mm Tris-HCl, 89 mmboric acid, and 2 mm EDTA, pH 8.8, and visualized by autoradiography. To quantify the product, the gel was scanned on a PhosphorImager 450 and the data were analyzed using ImageQuant software. dRP lyase activity was performed in a reaction mixture (10 μl) containing 50 mm Hepes, pH 7.4, 10 mm MgCl2, 2 mm DTT, and 20 nm pre-incised 32P-labeled normal AP-DNA or MX-adducted DNA. The reaction was initiated by adding β-pol (0–25 nm as indicated) and incubated at 37 °C for 15 min. The reaction was terminated by transfer to 0–1 °C, and the DNA product was stabilized by addition of NaBH4 as described above. The stabilized DNA products were processed as described above and visualized by autoradiography. The excision reaction was reconstituted in a reaction mixture (10 μl) that contained 50 mm Hepes, pH 7.4, 2 mm DTT, 10 mm MgCl2, 0.5 mm EDTA, 2 mm ATP, 20 μm each dATP, dGTP, dCTP, and TTP, and APE-preincised 32P-labeled duplex oligonucleotide substrate (20 nm). The reaction was initiated by adding 20 nm FEN1 and β-pol, as indicated in the figure legend. Incubation was at 37 °C for 30 min. The reaction was terminated by transfer to 0–1 °C, and the DNA product was stabilized by addition of NaBH4 as described above. The stabilized DNA product was recovered by ethanol precipitation, separated by electrophoresis, and visualized by autoradiography. Cytotoxicity was determined by growth inhibition assays. It has been shown previously that SV40-transformed mouse embryonic fibroblasts grow logarithmically and at similar rates under the conditions of the assay (6.Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (794) Google Scholar). Cells were seeded at a density of 40,000 cells/well in six-well dishes. The following day, they were exposed for 1 h to a range of concentrations of MMS or MNU in growth medium without hygromycin. MMS was dissolved directly in the medium. A stock solution of MNU was freshly-prepared in dimethyl sulfoxide and dissolved in medium at the time of the experiment. After 1 h, the cells were washed with Hanks' balanced salt solution and fresh medium was added. For UV irradiation (254 nm), cell monolayers were washed twice with Hanks' balanced salt solution before UV exposure (0–20 J/m2) in a Stratalinker model 1800 (Stratagene, La Jolla, CA), followed by addition of growth medium. Dishes were incubated for 4–5 days at 34 °C in a 10% CO2 incubator until untreated control cells were approximately 80% confluent. Cells (triplicate wells for each drug concentration) were counted by a cell lysis procedure (30.Butler W.B. Anal. Biochem. 1984; 141: 70-73Crossref PubMed Scopus (119) Google Scholar), and results were expressed as the number of cells in drug-treated wells relative to cells in control wells (% control growth). The IC50 and IC90 values (defined as the concentration of agent required for 50% or 90% growth inhibition compared with untreated controls) were determined from concentration-percentage of growth inhibition curves. Cytotoxicity studies were also conducted in the presence of MX. A stock solution of MX (1–5 m in phosphate-buffered saline) was prepared immediately before use and NaOH added to achieve neutral pH. MX stock solution was added to the volume of medium required for the experiment, and the pH was re-adjusted to 7.2 by further addition of NaOH. Dilutions of MMS or MNU were prepared in the MX-containing medium, and cells were dosed as described above. In certain experiments, cells were further incubated in MX-medium for up to 7 h following the 1-h alkylating agent exposure. We used a plasmid-based uracil-containing DNA substrate (see “Experimental Procedures”) that allows simultaneous quantification of repair by the single-nucleotide and long patch subpathways of BER. Conditions were standardized to measure the efficiency (k cat/K m,DNA) of BER and involved using a limiting concentration of DNA and saturating or near-saturating concentrations of dNTP for both single-nucleotide and long patch BER subpathways. Restriction enzyme and electrophoretic analysis of BER products formed with [α-32P]dCTP as the labeled nucleoside triphosphate indicated that the level of dCMP incorporation by wild-type cell extract into the first nucleotide of the repair patch (SphI + NcoI fragment) was similar to dCMP incorporation into the first two nucleotides of the repair patch (HaeIII fragment; Fig.1 A). In the HaeIII fragment, the ratio of dCMP incorporation (first and second nucleotide):dAMP incorporation (third nucleotide) was approximately 10:1 (data not shown). Thus, as expected from earlier work (11.Fortini P. Pascucci B. Parlanti E. Sobol R.W. Wilson S.H. Dogliotti E. Biochemistry. 1998; 37: 3575-3580Crossref PubMed Scopus (200) Google Scholar), single-nucleotide BER of uracil-DNA was predominant over long patch BER in a wild-type cell extract. In the experiment shown in Fig. 1 B, it was found that the combined short and long patch BER activity of the wild-type extract (incorporation of labeled dCTP into position 1 and 2 of the repair patch) as well as long patch BER activity (incorporation of labeled dATP into position 3) were both partially inhibited by a neutralizing antibody to β-pol. Non-immune serum from the same rabbit was used in control incubations and did not inhibit single-nucleotide or long patch BER activity (data not shown). These results indicate clearly that a portion of long patch BER activity measured in the wild-type cell extract is dependent upon β-pol (≈75%). Taken together, these results indicate that in addition to a role in single-nucleotide BER, β-pol also participates in long patch BER in mouse fibroblast extracts. The primary amine of MX is capable of reacting with the aldehydic C1′ atom of the abasic site, as illustrated in Scheme FS1. The Schiff base intermediate produced after MX attack on the C1′ atom spontaneously resolves into the stably adducted sugar molecule. Therefore, MX treatment will render the abasic site refractory to the β-elimination step involved in the dRP lyase activity of β-pol. This property of the MX-adducted abasic site DNA is confirmed by the experiments illustrated in Fig. 2. Using the duplex oligonucleotide substrates shown in Fig. 2 A, the dRP lyase activity of β-pol could be detected on a APE-cleaved normal AP site, resulting in a 29-mer product (Fig. 2 B,lanes 5 and 6), but not on a pre-incised MX-adducted AP site (lanes 2 and3). We conclude that MX is capable of chemical deletion of single-nucleotide BER that depends upon elimination of the dRP moiety in the
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