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• W2023356259 abstract "DNA alkylation damage is primarily repaired by the base excision repair (BER) machinery in mammalian cells. In repair of the N-alkylated purine base lesion, for example, alkyl adenine DNA glycosylase (Aag) recognizes and removes the base, and DNA polymerase β (β-pol) contributes the gap tailoring and DNA synthesis steps. It is the loss of β-pol-mediated 5′-deoxyribose phosphate removal that renders mouse fibroblasts alkylation-hypersensitive. Here we report that the hypersensitivity of β-pol-deficient cells after methyl methanesulfonate-induced alkylation damage is wholly dependent upon glycosylase-mediated initiation of repair, indicating that alkylated base lesions themselves are tolerated in these cells and demonstrate that β-pol protects against accumulation of toxic BER intermediates. Further, we find that these intermediates are initially tolerated in vivo by a second repair pathway, homologous recombination, inducing an increase in sister chromatid exchange events. If left unresolved, these BER intermediates trigger a rapid block in DNA synthesis and cytotoxicity. Surprisingly, both the cytotoxic and genotoxic signals are independent of both the p53 response and mismatch DNA repair pathways, demonstrating that p53 is not required for a functional BER pathway, that the observed damage response is not part of the p53 response network, and that the BER intermediate-induced cytotoxic and genotoxic effects are distinct from the mechanism engaged in response to mismatch repair signaling. These studies demonstrate that, although base damage is repaired by the BER pathway, incomplete BER intermediates are shuttled into the homologous recombination pathway, suggesting possible coordination between BER and the recombination machinery. DNA alkylation damage is primarily repaired by the base excision repair (BER) machinery in mammalian cells. In repair of the N-alkylated purine base lesion, for example, alkyl adenine DNA glycosylase (Aag) recognizes and removes the base, and DNA polymerase β (β-pol) contributes the gap tailoring and DNA synthesis steps. It is the loss of β-pol-mediated 5′-deoxyribose phosphate removal that renders mouse fibroblasts alkylation-hypersensitive. Here we report that the hypersensitivity of β-pol-deficient cells after methyl methanesulfonate-induced alkylation damage is wholly dependent upon glycosylase-mediated initiation of repair, indicating that alkylated base lesions themselves are tolerated in these cells and demonstrate that β-pol protects against accumulation of toxic BER intermediates. Further, we find that these intermediates are initially tolerated in vivo by a second repair pathway, homologous recombination, inducing an increase in sister chromatid exchange events. If left unresolved, these BER intermediates trigger a rapid block in DNA synthesis and cytotoxicity. Surprisingly, both the cytotoxic and genotoxic signals are independent of both the p53 response and mismatch DNA repair pathways, demonstrating that p53 is not required for a functional BER pathway, that the observed damage response is not part of the p53 response network, and that the BER intermediate-induced cytotoxic and genotoxic effects are distinct from the mechanism engaged in response to mismatch repair signaling. These studies demonstrate that, although base damage is repaired by the BER pathway, incomplete BER intermediates are shuttled into the homologous recombination pathway, suggesting possible coordination between BER and the recombination machinery. Cancer, or tumor development, is generally the result of one or more genetic changes in critical growth or cellular maintenance genes. Such changes may be inherited or may occur from environmental exposure (1Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22406) Google Scholar, 2Hoeijmakers J.H. Nature. 2001; 411: 366-374Crossref PubMed Scopus (3149) Google Scholar). In many cases, these genetic changes induce a “mutator” phenotype due to sequence changes in DNA repair or DNA damage checkpoint genes, among others (3Loeb L.A. Cancer Res. 1991; 51: 3075-3079PubMed Google Scholar). The importance of maintaining the structural and informational integrity of the genome is further highlighted by the plethora of DNA repair and DNA damage checkpoint pathways (2Hoeijmakers J.H. Nature. 2001; 411: 366-374Crossref PubMed Scopus (3149) Google Scholar). Consequently, it is important to understand the diverse cellular systems for DNA damage repair available for protection from an enormous array of DNA lesions (4Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1279) Google Scholar). The base excision repair (BER) 1The abbreviations used are: BER, base excision repair; APendo, apurinic/apyrimidinic endonuclease; β-pol, DNA polymerase β; 5′-dRP, 5′-deoxyribosephosphate; MMS, methyl methanesulfonate; Aag, alkyladenine DNA glycosylase; BrdUrd, bromodeoxyuridine; SCE, sister chromatid exchange; MNNG, N-methyl-N′-nitro-N-nitrosoguanidine; 3-MeA, 3-methyladenine; ES, embryonic stem; hAAG, human alkyladenine DNA glycosylase; MEF, mouse embryonic fibroblast; PBS, phosphate-buffered saline; wt, wild type; GFP, green fluorescent protein. pathway is considered the predominant DNA repair system in mammalian cells for eliminating small DNA lesions generated either exogenously or endogenously at DNA bases (4Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1279) Google Scholar, 5Seeberg E. Eide L. Bjoras M. Trends Biochem. Sci. 1995; 20: 391-397Abstract Full Text PDF PubMed Scopus (470) Google Scholar, 6Wilson S.H. Mutat. Res. 1998; 407: 203-215Crossref PubMed Scopus (265) Google Scholar). Such DNA damage can be caused by exposure to environmental agents or by normal cellular metabolic processes that produce alkylating molecules, reactive oxygen species, and other reactive metabolites capable of modifying DNA. In the mammalian BER pathway, the damaged base residue is removed by a lesion-specific DNA glycosylase. Subsequently, the resulting abasic site is recognized by apurinic/apyrimidinic endonuclease (APendo), which incises the damaged strand, leaving a single-nucleotide gap with 3′-OH and 5′-deoxyribose phosphate (5′-dRP) groups at the margins. A DNA polymerase β (β-pol)-mediated DNA synthesis step extends from the 3′-OH (7Singhal R.K. Prasad R. Wilson S.H. J. Biol. Chem. 1995; 270: 949-957Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 8Sobol R.W. Wilson S.H. Prog. Nucleic Acids Res. Mol. Biol. 2001; 68: 57-74Crossref PubMed Google Scholar, 9Wilson S.H. Sobol R.W. Beard W.A. Horton J.K. Prasad R. Vande Berg B.J. Cold Spring Harbor Symp. Quant. Biol. 2001; 65: 143-155Crossref Scopus (48) Google Scholar), and the 5′-dRP group is removed by the 5′-dRP lyase activity of β-pol (10Piersen C.E. Prasad R. Wilson S.H. Lloyd R.S. J. Biol. Chem. 1996; 271: 17811-17815Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 11Srivastava 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 (348) Google Scholar, 12Matsumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (649) Google Scholar, 13Prasad R. Beard W.A. Chyan J.Y. Maciejewski M.W. Mullen G.P. Wilson S.H. J. Biol. Chem. 1998; 273: 11121-11126Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 14Prasad R. Beard W.A. Strauss P.R. Wilson S.H. J. Biol. Chem. 1998; 273: 15263-15270Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). DNA ligase I or a complex of DNA ligase III and x-ray cross-complementing factor 1 conducts the final, nick sealing, step in the pathway. In addition, several proteins have been observed to form functional partnerships with these BER proteins, including p53, poly(ADP-ribose) polymerase, p300, and proliferating cell nuclear antigen (15Lavrik O.I. Prasad R. Sobol R.W. Horton J.K. Ackerman E.J. Wilson S.H. J. Biol. Chem. 2001; 276: 25541-25548Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 16Kedar P.S. Kim S.J. Robertson A. Hou E. Prasad R. Horton J.K. Wilson S.H. J. Biol. Chem. 2002; 277: 31115-31123Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 17Hasan S. El-Andaloussi N. Hardeland U. Hassa P.O. Burki C. Imhof R. Schar P. Hottiger M.O. Mol. Cell. 2002; 10: 1213-1222Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 18Zhou J. Ahn J. Wilson S.H. Prives C. EMBO J. 2001; 20: 914-923Crossref PubMed Scopus (278) Google Scholar). In vitro studies suggested a role for β-pol in varying types of DNA repair (19Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (263) Google Scholar). Transgenic mice with a homozygous null mutation in the β-pol gene are nonviable after birth, thereby preventing studies on the in vivo function of β-pol (20Gu H. Marth J.D. Orban P.C. Mossmann H. Rajewsky K. Science. 1994; 265: 103-106Crossref PubMed Scopus (1164) Google Scholar). However, we utilized β-pol (+/–) mice to establish embryonic fibroblast (MEF) cell lines homozygous for a null mutation in the β-pol gene (21Sobol 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 (784) Google Scholar). β-pol (–/–) cells are normal in viability and growth characteristics but are more sensitive to monofunctional alkylating agents such as methyl methanesulfonate (MMS) than wild type cells (21Sobol 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 (784) Google Scholar). β-pol possesses both polymerase and 5′-dRP lyase activity, but we found previously that only the 5′-dRP lyase activity of β-pol is essential for resistance to MMS and that β-pol appears to be the major, if not only, protein capable of efficiently removing 5′-dRP groups formed as BER intermediates (22Sobol R.W. Prasad R. Evenski A. Baker A. Yang X.P. Horton J.K. Wilson S.H. Nature. 2000; 405: 807-810Crossref PubMed Scopus (299) Google Scholar). However, these earlier studies did not determine the source of these repair intermediates, and it was not yet determined if the 5′-dRP groups were formed as a result of BER initiation by a lesion-specific glycosylase. Further, no study to date has begun to unravel the mechanism of cytoxicity induced by the BER intermediate 5′-dRP. In this report, we describe the development and characterization of a series of single and double knockout cell lines and mouse models to detail the cytotoxic and genotoxic effects of both alkylated base lesions and the resultant BER intermediate 5′-dRP. We show that 5′-dRP formation (the lethal lesion causing the MMS-sensitive phenotype of β-pol (–/–) cells) is critically dependent on the alkyladenine DNA glycosylase (Aag), known to remove 3-methyladenine (3-MeA), 7-methylguanine, and other alkylated bases from DNA. Surprisingly, the unrepaired N-alkylated purine base lesions are well tolerated in these cells. Interestingly, our results suggest that BER intermediates are associated with an increase in homologous recombination and eventually trigger a block in DNA synthesis. Both the cytotoxic and genotoxic effects in these cells are independent of p53 and the mismatch DNA repair pathway. These studies suggest a possible coordination between BER and the recombination machinery. Mice and Cells—All breeding was conducted at the NIEHS animal facility following National Institutes of Health, Institutional Animal Care and Use Committee and the Association for Assessment and Accreditation of Laboratory Animal Care approved protocols. The β-pol (+/–) mice and the Aag (–/–) mice have been described previously (20Gu H. Marth J.D. Orban P.C. Mossmann H. Rajewsky K. Science. 1994; 265: 103-106Crossref PubMed Scopus (1164) Google Scholar, 23Engelward B.P. Weeda G. Wyatt M.D. Broekhof J.L. de Wit J. Donker I. Allan J.M. Gold B. Hoeijmakers J.H. Samson L.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13087-13092Crossref PubMed Scopus (206) Google Scholar). The APendo (+/–) mice were generously provided by T. Curran (St. Jude Children's Research Hospital) (24Xanthoudakis S. Smeyne R.J. Wallace J.D. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8919-8923Crossref PubMed Scopus (438) Google Scholar). The p53 (+/–) mice were generously provided by G. Lozano (M.D. Anderson Cancer Center) (25Jacks T. Remington L. Williams B.O. Schmitt E.M. Halachmi S. Bronson R.T. Weinberg R.A. Curr. Biol. 1994; 4: 1-7Abstract Full Text Full Text PDF PubMed Scopus (1740) Google Scholar). The PMS-2 (+/–) mice were generously provided by P. M. Glazer (Yale University) (26Narayanan L. Fritzell J.A. Baker S.M. Liskay R.M. Glazer P.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3122-3127Crossref PubMed Scopus (129) Google Scholar). Wild type, β-pol (–/–), Aag (–/–), β-pol (–/–)/Aag (–/–), p53 (–/–), β-pol (–/–)/p53 (–/–), PMS-2 (–/–), and β-pol (–/–)/PMS-2 (–/–) primary MEFs were isolated from 14.5-day-old embryos and, where indicated, transformed by SV40 T-antigen expression, as previously described (21Sobol 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 (784) Google Scholar). Genotyping and reverse transcriptase-PCR protocols are available upon request. The MMP-GFP and MMP-hAAG retroviral vectors were developed in conjunction with the Harvard University gene therapy initiative (available on the World Wide Web at hgti.med.harvard.edu/PlasmidRepository.php3). MEFs were cultured at 37 °C in a humidified incubator equilibrated with 10% CO2 in DMEM supplemented with 10% fetal bovine serum, penicillin (50 units/ml), streptomycin (50 μg/ml), and Glutamax-I (4 mm) as described (21Sobol 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 (784) Google Scholar, 22Sobol R.W. Prasad R. Evenski A. Baker A. Yang X.P. Horton J.K. Wilson S.H. Nature. 2000; 405: 807-810Crossref PubMed Scopus (299) Google Scholar, 28Sobol R.W. Watson D.E. Nakamura J. Yakes F.M. Hou E. Horton J.K. Ladapo J. Van Houten B. Swenberg J.A. Tindall K.R. Samson L.D. Wilson S.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6860-6865Crossref PubMed Scopus (74) Google Scholar). Gap-filling Assay—Whole cell extracts were assayed using a single-nucleotide gap-filling assay. Extracts were incubated in buffer containing 50 mm Hepes, pH 7.8, 2 mm EDTA, 1 mm dithiothreitol, 10 mm MgCl2, 100 mm KCl, 330 nm [α-32P]dCTP, and 100 nm double-stranded oligonucleotide containing a single uracil (U) residue: 5′-GCC CTG CAG GTC GAU TCT AGA GGA TCC CCG GGT AC-3′. The reaction was incubated for 5 min at 37 °C and analyzed by electrophoresis in a 16% polyacrylamide gel (7 m urea, TBE). Gels were fixed, dried and autoradiographed. Alkyladenine glycosylase assay—Aag activity was measured as described previously (23Engelward B.P. Weeda G. Wyatt M.D. Broekhof J.L. de Wit J. Donker I. Allan J.M. Gold B. Hoeijmakers J.H. Samson L.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13087-13092Crossref PubMed Scopus (206) Google Scholar) using a double-stranded oligonucleotide substrate containing a single etheno-adenine (eA) lesion (29Kartalou M. Samson L.D. Essigmann J.M. Biochemistry. 2000; 39: 8032-8038Crossref PubMed Scopus (37) Google Scholar): 5′-GCA ATC TAG CCA eAGT CGA TGT ATG C-3′. The reaction was incubated for 20 min at 37 °C and analyzed by electrophoresis in a 20% polyacrylamide gel (7 m urea, TBE). Gels were fixed, dried, and autoradiographed. Cytotoxicity Assays—Cytotoxicity was determined by growth inhibition assays as described previously (22Sobol R.W. Prasad R. Evenski A. Baker A. Yang X.P. Horton J.K. Wilson S.H. Nature. 2000; 405: 807-810Crossref PubMed Scopus (299) Google Scholar). Briefly, cells were exposed to the DNA-damaging agent, MMS or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), 24 h after seeding (40,000 cells/well) in 6-well dishes. Cells (triplicate wells) were then treated for 1 h at 37 °C in a 10% CO2 incubator with serial dilutions of mutagen in growth medium. For each experiment, cells were counted after 3 days (untreated cells are found to be <80% confluent). Cell numbers will be determined by a cell lysis protocol. Results are the mean of at least two separate experiments. Percentage of control growth is as follows: (number of treated cells)/(number of control cells)·100. Flow Cytometric Cell Cycle Analysis—Cell cycle and DNA synthesis was analyzed simultaneously by staining with propidium iodide and incorporation of bromodeoxyuridine (BrdUrd) with some modifications (30Kastan M.B. Onyekwere O. Sidransky D. Vogelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar). Briefly, cells were seeded in 100-mm dishes at a density of 327,000 cells/dish. (equivalent to the cell density used in the cytotoxicity experiments). The following day, cells were treated for 1 h with MMS. At 2 or 8 h after MMS treatment (as indicated), 10 μm BrdUrd (Sigma) was added to the dishes for 30 min to pulse-label the cells. Cells were then washed with PBS, harvested by trypsinization, and washed a second time with PBS. The cell pellet obtained after centrifugation was resuspended in 100 μl of cold PBS, and the cells were dropped slowly into 70% ethanol and allowed to fix at 4 °C overnight. The samples were washed, suspended in 2 n HCl containing 0.5% Triton X-100, and incubated for 30 min at room temperature to denature the DNA. The cell samples were pelleted, resuspended in 0.1 m sodium borate (pH 8.5) to neutralize the acid and then washed with PBS. Cells were then incubated at 4 °C overnight with 20 μl of anti-BrdUrd-fluorescein isothiocyanate-conjugated antibody (BD Biosciences) in PBS containing 0.5% Tween 20 and 1% bovine serum albumin and 5 μl of 10 mg/ml RNase (Sigma) stock solution. The following day, the cells were pelleted, washed with PBS, and resuspended in 1 ml of PBS containing 5 μg/ml propidium iodide (Sigma). The samples were analyzed by flow cytometry using Cell Quest software (BD Biosciences). Cell cycle populations are designated G0/G1 (2 n DNA content with no BrdUrd incorporation), S (variable DNA content with BrdUrd incorporation), and G2/M (4 n DNA content without BrdUrd incorporation). Sister Chromatid Exchange Assay—For sister chomatid exchange (SCE) measurements, 1 × 106 cells were seeded onto 75-cm2 tissue culture dishes 8 h before drug treatment. The cells were treated with MMS in complete McCoy's 5A medium (Invitrogen) supplemented with 10 μm BrdUrd for 1 h at 37°C in 5% CO2. Following drug treatment, cells were incubated in McCoy's 5A medium supplemented with 10 μm BrdUrd for 20 h. Colcemid (0.1 μg/ml; Invitrogen) was included for the last 2 h of incubation, and the cells were subsequently harvested by mitotic shake-off, resuspended, and incubated for 15 min at 37 °C in hypotonic solution (0.2% potassium chloride, 0.2% sodium citrate, and 10% fetal bovine serum) and then fixed in Carnoy's solution. To produce “harlequin” chromosomes, a modified fluorescence plus Giemsa technique was used (31Perry P. Wolff S. Nature. 1974; 251: 156-158Crossref PubMed Scopus (3026) Google Scholar). Slides were stained in Hoechst 33258 (5 μg/ml) for 20 min, mounted in 0.067 m Sorensen's buffer with a coverslip, and exposed to a General Electric 15-watt black light bulb at 65 °C for 20 min. Slides were then heated at 65 °C in 20× SSC for 20 min, rinsed, and stained in a 5% Giesma solution in 0.067 m Sorensen's buffer. Twenty second-division metaphase spreads were counted per data point. Aag Is Required to Initiate BER of MMS- and MNNG-induced Lesions to Generate the Toxic 5′-dRP Repair Intermediate—To study the cytotoxicity and genotoxicity of both alkylated DNA bases and BER repair intermediates, we developed a set of isogenic MEF cell lines with homozygous null mutations in the Aag gene, in the β-pol gene or in both the Aag and β-pol genes (Fig. 1). To ensure that no significant backup or compensatory enzymatic activity was present, extracts from each were analyzed for Aag-specific activity (Fig. 2a) and β-pol-specific BER gap-filling activity (Fig. 2b). Wild type and β-pol (–/–) cells showed the expected levels of Aag activity, with little or no Aag-specific activity in the Aag (–/–) and β-pol (–/–)/Aag (–/–) cells (Fig. 2a), whereas each had similar levels of uracil-DNA glycosylase activity (data not shown). Conversely, the wild type and Aag (–/–) cells reported robust β-pol-specific BER activity with little or no activity seen in extracts from β-pol (–/–) or β-pol (–/–)/Aag (–/–) cells (Fig. 2b).Fig. 2Evidence that methylation damage is noncytotoxic, yet Aag induces the formation of cytotoxic β-pol substrates. a, Aag activity. Whole cell extract (20 μg) was analyzed for Aag activity using a double-stranded DNA oligonucleotide (26-bp) containing a single eA lesion (23Engelward B.P. Weeda G. Wyatt M.D. Broekhof J.L. de Wit J. Donker I. Allan J.M. Gold B. Hoeijmakers J.H. Samson L.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13087-13092Crossref PubMed Scopus (206) Google Scholar, 29Kartalou M. Samson L.D. Essigmann J.M. Biochemistry. 2000; 39: 8032-8038Crossref PubMed Scopus (37) Google Scholar). Filled bars correspond to the parental cell lines, whereas the open bars (*) correspond to stable cell clones transduced with a retrovirus expressing hAAG. b, β-pol activity. Whole cell extract (20 μg) was analyzed for β-pol activity using a double-stranded DNA oligonucleotide (34-bp) containing a single uracil residue (21Sobol 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 (784) Google Scholar). Lane 1, wild type; lane 2, β-pol (–/–); lane 3, Aag (–/–); lane 4, β-pol (–/–)/Aag (–/–). The arrow points to the expected DNA synthesis product. Sensitivity of fibroblast cell lines to MMS. Shown are survival curves in the presence of MMS of wild type cells (filled circles) versus β-pol (–/–) cells (filled triangles) (c); wild type cells (filled circles) versus Aag (–/–) cells (filled squares) (d); β-pol (–/–) cells (filled triangles) versus β-pol (–/–)/Aag (–/–) cells (filled diamonds) (e); β-pol (–/–)[+GFP] cells (filled triangles) versus β-pol (–/–)[+hAAG] cells (open triangles) (f); and β-pol (–/–)/Aag (–/–)[+GFP] cells (filled diamonds) versus β-pol (–/–)/Aag (–/–)[+hAAG] cells (open diamonds) (g). The arrows indicate the shift of the curve due to the genetic loss of β-pol (c), the genetic loss of Aag (e), or the overexpression of hAAG (f and g). Values presented are the means ± S.D. of two independent experiments. Within each experiment, means are calculated from triplicate values.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The cytotoxicity of methylating agent-induced DNA base damage is increased in both Aag (–/–) embryonic stem (ES) cells (32Engelward B.P. Dreslin A. Christensen J. Huszar D. Kurahara C. Samson L. EMBO J. 1996; 15: 945-952Crossref PubMed Scopus (184) Google Scholar) and β-pol (–/–) MEFs (21Sobol 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 (784) Google Scholar), implying that both methylated bases and BER intermediates are cytotoxic. We therefore compared the cytotoxicity of MMS in wild type and isogenic β-pol (–/–) (Fig. 2c) and Aag (–/–) MEFs (Fig. 2d). Consistent with the known phenotype of these cells and the hypothesis that the 5′-dRP lyase activity of β-pol is essential for reversing MMS-induced cytotoxicity (22Sobol R.W. Prasad R. Evenski A. Baker A. Yang X.P. Horton J.K. Wilson S.H. Nature. 2000; 405: 807-810Crossref PubMed Scopus (299) Google Scholar), β-pol (–/–) MEFs were more sensitive to MMS than wild type MEFs, as expected (Fig. 2c). We next tested Aag (–/–) MEFs to directly evaluate the cytotoxicity of Aag substrates, since MMS causes a significant buildup of the principal Aag substrate 3-MeA in Aag (–/–) ES cells (33Engelward B.P. Allan J.M. Dreslin A.J. Kelly J.D. Wu M.M. Gold B. Samson L.D. J. Biol. Chem. 1998; 273: 5412-5418Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 34Smith S.A. Engelward B.P. Nucleic Acids Res. 2000; 28: 3294-3300Crossref PubMed Scopus (46) Google Scholar). Surprisingly, Aag substrates generated by MMS exposure conferred no increase in cytotoxicity (Fig. 2d). This finding suggests that lesion avoidance mechanisms are more robust in MEFs than ES cells (see below). The lack of MMS-induced cytotoxicity allowed us to characterize the cytotoxic and genotoxic effects of BER intermediates in these cells. Next, we compared β-pol (–/–) cells with β-pol (–/–)/Aag (–/–) cells for MMS-induced cytotoxicity (Fig. 2e). We found that the MMS-sensitive phenotype of β-pol (–/–) cells is critically dependent on Aag activity, because β-pol (–/–)/Aag (–/–) cells were much more resistant to MMS than β-pol (–/–) cells (Fig. 2e). β-pol (–/–) MEFs have also been reported to be hypersensitive to alkylation damage caused by MNNG (21Sobol 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 (784) Google Scholar). Using the same set of four isogenic cell lines, we compared the cytotoxicity profile induced by MNNG to determine whether this induced cytotoxic response is also mediated by repair intermediates initiated by Aag. Identical to the results for MMS treatment, β-pol (–/–) MEFs but not Aag (–/–) MEFs were hypersensitive to MNNG, and the genetic loss of Aag prevented the formation of repair intermediates that cause the cytotoxicity in the absence of β-pol (Fig. 3). It should be noted that all four cell lines exhibited similar cytotoxic profiles following UV exposure (data not shown). Therefore, both MMS and MNNG generate Aag substrates in DNA; if left unrepaired, these Aag substrates are not cytotoxic in these cells, yet accumulation of the 5′-dRP repair intermediate (formed following Aag-mediated lesion removal and APendo hydrolysis of the abasic site) induces a dramatic cytotoxic response. To further demonstrate that Aag-mediated lesion removal contributes to the generation of cytotoxic β-pol substrates, we transduced wild type, β-pol (–/–), Aag (–/–), and β-pol (–/–)/Aag (–/–) cells with the MMP retrovirus expressing either GFP or human alkyladenine DNA glycosylase (hAAG). To ensure uniform hAAG expression levels, individual clones were isolated and selected for similar AAG activity. As shown in Fig. 2a, all hAAG overexpressing lines had activity levels approaching 5 times that seen in wild type cells. Consistent with the model that Aag-mediated lesion removal is required for the formation of cytotoxic β-pol substrates, hAAG overexpression increased the MMS-induced cytotoxicity in a β-pol (–/–) background. As shown in Fig. 2f, β-pol (–/–)[+hAAG] cells were significantly more sensitive to MMS treatment than the control β-pol (–/–)[+GFP] cells. Further, hAAG overexpression in β-pol (–/–)/Aag (–/–) cells restored the MMS-induced hypersensitivity that is the hallmark of the β-pol (–/–) phenotype (21Sobol 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 (784) Google Scholar, 22Sobol R.W. Prasad R. Evenski A. Baker A. Yang X.P. Horton J.K. Wilson S.H. Nature. 2000; 405: 807-810Crossref PubMed Scopus (299) Google Scholar) (Fig. 2g). Aag Deficiency Does Not Rescue the Embryonic or Perinatal Lethality of the APendo( – / – ) and the β-pol( – / – ) Mutation in Mice—The cellular phenotype associated with a genetic loss of β-pol (i.e. a DNA damage-induced accumulation and cytotoxicity of the 5′-dRP lesion) (21Sobol 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 (784) Google Scholar, 22Sobol R.W. Prasad R. Evenski A. Baker A. Yang X.P. Horton J.K. Wilson S.H. Nature. 2000; 405: 807-810Crossref PubMed Scopus (299) Google Scholar), raised the question of whether the lethality of the β-pol (–/–) mutation in mice (20Gu H. Marth J.D. Orban P.C. Mossmann H. Rajewsky K. Science. 1994; 265: 103-106Crossref PubMed Scopus (1164) Google Scholar, 35Esposito G. Texido G. Betz U.A. Gu H. Muller W. Klein U. Rajewsky K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1166-1171Crossref PubMed Scopus (84) Google Scholar, 36Sugo N. Aratani Y. Nagashima Y. Kubota Y. Koyama H. EMBO J. 2000; 19: 1397-1404Crossref PubMed Google Scholar) is mediated by a similar phenomenon. Clearly, spontaneous generation of Aag substrates does not lead to lethality in mice, since Aag (–/–) mice are viable and are without an obvious phenotype (23Engelward B.P. Weeda G. Wyatt M.D. Broekhof J.L. de Wit J. Donker I. Allan J.M. Gold B. Hoeijmakers J.H. Samson L.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13087-13092Crossref PubMed Scopus (206) Google Scholar, 37Hang B. Singer B. Margison G.P. Elder R.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12869-12874Crossref PubMed Scopus (127) Google Scholar). We therefore bred a colony of β-pol (+/–)/Aag (–/–) mice to determine if genetic loss of Aag would rescue the β-pol (–/–)-associated lethality. As shown in Table I, the Aag (–/–) mutation did not rescue the β-pol( –/–) lethality. At a minimum, this reveals that the β-pol (–/–) lethal phenotype is not due to Aag-initiated repair intermediates. Similar studies with APendo (+/–)/Aag (–/–) mice suggest that the APendo (–/–) lethal phenotype is also not due to Aag-initiated repair intermediates. It remains to be determined whether genetic loss of any or all of the damage-spec" @default.
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