Base Excision Repair Intermediates Induce p53-independent Cytotoxic and Genotoxic Responses

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. 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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 formed as a result of BER initiation by a lesion-specific glycosylase. Further, to to the mechanism of by the BER In we the and of a of and cell lines and mouse to the cytotoxic and genotoxic effects of both alkylated base lesions and the BER that 5′-dRP the phenotype of β-pol (–/–) is dependent on the alkyladenine DNA glycosylase to and other alkylated bases from DNA. Surprisingly, the N-alkylated purine base lesions are tolerated in these cells. that BER intermediates are with an increase in homologous recombination and trigger a block in DNA the cytotoxic and genotoxic effects in these cells are independent of p53 and the mismatch DNA repair pathway. These studies a possible coordination between BER and the recombination machinery. and was at the of and and the for and of The β-pol (+/–) mice and the (–/–) mice have been previously (20Gu H. Marth J.D. Orban P.C. Mossmann H. Rajewsky K. Science. 1994; 265: 103-106Crossref PubMed Scopus (1164) Google Scholar, G. J. J.H. Sci. U. S. A. PubMed Scopus Google Scholar). The (+/–) mice by T. S. J.D. T. Sci. U. 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The cell in to the and with at with of in and and of The the cells with PBS, and in 1 of The by are DNA with DNA with and DNA sister exchange 1 cells The cells with MMS in with for 1 at in cells in with for was for the of and the cells by and for at in and and in produce a was used P. S. Nature. PubMed Scopus Google Scholar). in for in with a and to a at for at in for and in a in to BER of and to the 5′-dRP the and of both alkylated DNA bases and BER repair intermediates, we a of cell lines with homozygous null in the in the β-pol gene or in both the and β-pol that or activity was from for activity and BER activity type and β-pol (–/–) cells the of activity, with or activity in the (–/–) and β-pol (–/–) cells of glycosylase activity not the wild type and (–/–) cells BER activity with or activity in from β-pol (–/–) or β-pol (–/–) cells that damage is yet the of cytotoxic β-pol cell was for activity a DNA a G. J. J.H. Sci. U. S. A. 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DNA from cell was and by to either the or null to the β-pol and The of the the of either the or null as of fibroblast cell lines to in the of MMS of wild type cells β-pol (–/–) cells (–/–) cells and β-pol (–/–) cells The the of the due to the genetic loss of 5′-dRP by of the may be by homologous recombination of E. G. M. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, Science. 2002; PubMed Scopus Google Scholar). β-pol or BER intermediates (5′-dRP) block we determined these BER intermediates can induce toxic of MMS to we the genotoxic of both base damage and BER intermediates by the of in and MMS of wild and β-pol (–/–) generated a increase in events. However, BER intermediates in β-pol (–/–) generated more indicating that 5′-dRP lesions can homologous to the in the β-pol (–/–) cells activity to to the than wild type of Further, of in β-pol (–/–) cells the increase in and of MMS as determined by are of β-pol (–/–) cells β-pol (–/–) cells with MMS for 1 β-pol cells and β-pol cells with In the to an of In we further the that BER intermediates can be in and of in the of to the MMS hypersensitivity observed for β-pol cells is dependent upon initiation of repair pathway by the DNA glycosylase. The that the alkylating (MMS) hypersensitivity of the β-pol (–/–) cell was not observed in the of the of hypersensitivity in cell system the of and the of the are with the that the BER is more toxic to these cells than the alkylated DNA resulting from the MMS these cells are to DNA lesions a cell in the null cell the cell DNA synthesis but to DNA synthesis after a The mechanism of and is at the However, a the is or by a DNA the 5′-dRP β-pol (–/–) cells of DNA synthesis and an of DNA However, to the block in is from several have a of MMS upon example, and found that cells from (–/–) mice than cells from wild type other groups found that (–/–) cells and more or than G. J. J.H. Sci. U. S. A. PubMed Scopus Google Scholar, G.P. Sci. U. S. A. PubMed Scopus Google Scholar, G.P. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar, Cancer Res. 2002; Google Scholar). 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