HUMAN DNA REPAIR ENZYME O6-METHYLGUANINEDNA METHYLTRANSFERASE IN CELLULAR REGULATION UPON DNA DAMAGE OH HUE KIAN (B.Sc (Hons.), University of Leeds) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2004 Acknowledgements I am most grateful to my supervisor, Associate Professor Benjamin F.L.Li, for his constant support, guidance, encouragement and patience throughout my graduate studies I would like to thank Associate Professor Uttam Surana and Associate Professor Edward Manser, members of my supervisory committee Their comments, advices and discussions have been a great help to me during the progress of my work I thank the past and present members in our laboratory for making it a great place to work in Many thanks to Siew Wee, Tsui Han, Rahmen and Lydia for their encouragement and their support and many others for sharing reagents and for their help in many ways Special thanks to Hannah-Claire for her help in making this thesis possible and for her friendship Finally, my heartfelt gratitude goes to my family for their constant support i Table of Contents Acknowledgements i Table of Contents ii List of Publications vi Abbreviations vii Summary ix Chapter Introduction 1.1 Types of DNA Damages 1.1.1 DNA instability 1.1.2 Oxidative and alkylation damages 1.1.3 Irradiation Damage 1.1.4 Mutagenic Chemicals 1.2 DNA Repair Pathways .5 1.2.1 Base Excision Repair (BER) 1.2.2 Nucleotide Excision Repair (NER) 1.2.3 Mismatch Repair (MMR) 1.2.4 Double Strand Breaks Repair (DSB) 10 1.2.5 Direct Repair 13 1.3 Nuclear Hormone Receptor .16 1.3.1 The nuclear receptor superfamily 16 1.3.2 Structure/ domains of receptors 17 1.3.3 Receptor classifications 21 1.3.4 Transcriptional coregulators of NRs 23 1.3.4.1 Nuclear receptor co-activators 24 1.3.4.2 Co-integrators for NR-dependent transactivation 25 1.3.4.3 Nuclear receptor corepressor 27 1.3.4.4 The chromatin link 28 1.3.5 1.4 1.4.1 Function and regulation of Estrogen receptor 29 The MDM2/p53 pathway 34 MDM2 is oncogenic 34 ii 1.4.2 Stucture of MDM2 34 1.4.3 The tumour suppressor p53 36 1.4.4 MDM2 –p53 autoregulatory feedback pathway 38 1.4.4.1 MDM2 blocks p53 transcriptional activity 38 1.4.4.2 MDM2 role as ubiquitin E3 ligase 38 1.4.5 1.5 Regulation of MDM2-p53 loop 39 Aims of research 42 Chapter Materials and Methods .43 2.1 Cell Lines 43 2.2 Antibodies 43 2.3 Drugs, Chemicals and other reagents .44 2.4 Epitope-Mapping of Mab.3C7 44 2.5 Assay of MGMT Repair Activity 45 2.6 Protease V8 Digestion 45 2.7 Cell lysis and Western blot analyses (Immunoblotting) 45 2.8 In vitro binding of GST-(wt) and MGMT (K107L) to ERα 46 2.9 In vitro binding of GST-(wt) and GST-p53 to MBP-MDM2 47 2.10 Flow Cytometry 47 2.11 Transfection by Superfect® .47 2.12 Transfection by Calcium Phosphate precipitation procedure 48 2.13 ERE / GRE Reporter Assay .48 2.14 Mammalian Two-Hybrid Assay 49 2.15 Immunoprecipitation experiments .50 2.16 Drug/ionizing treatments (section 3.3) 50 2.17 Chromatin Immunoprecipitation Assay (ChIP) 51 Chapter Results 53 3.1 Conformational Change in MGMT Upon Active-Site Alkylation 56 3.1.1 Detection of R-MGMT by protease V8 assay using recombinant MGMT 56 3.1.2 Alkylating agents (SN1 and SN2) in the formation of R-MGMT 56 iii 3.1.3 Monoclonal antibody 3C7 preferentially immunoprecipitates RMGMT 61 3.1.4 Epitope Mapping of Mab 3C7 63 3.1.5 Characterization of mutant proteins with point mutation at the Mab 3C7 65 3.1.6 3.2 Discussions 67 Role of R-MGMT in the regulation of Estrogen receptor 73 3.2.1 Proteins homologous to the Mab3C7 epitope 73 3.2.2 Effect of R-MGMT on the growth of ER-expressing cells 73 3.2.3 Cell cycle arrest by 6BG and MeI is independent of p53 75 3.2.4 R-MGMT Interacts with Estrogen Receptor (ERα) in vitro 78 3.2.5 Interactions of endogenous R-MGMT and ERα 82 3.2.6 MGMT K107L mutant resembles R-MGMT-mediated G1 arrest in MCF7 cells 83 3.2.7 R-MGMT affects ER-mediated transcription complex 83 3.2.8 R-MGMT inhibits ERα transcriptional activity 87 3.2.9 Interaction and regulation of other nuclear receptor(s) by R-MGMT 87 3.2.10 Discussions 90 3.3 Role of MGMT and BRCA1 in the regulation of Estrogen receptor 95 3.3.1 The relationship between MGMT and ERα proteins 95 3.3.2 Antisense MGMT downregulates ERα expression and leads to cell cycle arrest 95 3.3.3 MGMT associates with ERα promoter 99 3.3.4 Regulation of ERα promoter by MGMT and BRCA1 upon DNA damages 102 3.3.4.1 Inter-relationships among MGMT, ERα and BRCA1 upon DNA damage 102 3.3.4.2 Phosphorylated BRCA1 displaces MGMT from ERα promoter upon exposure to oxidation damage 104 3.3.4.3 MGMT augments ERα-mediated transcription 108 3.3.5 3.4 Discussions 108 Role of R-MGMT in the p53-MDM2 regulatory pathway 113 3.4.1 Sequence homology between p53 and MGMT 113 3.4.2 Direct interaction between MGMT and MDM2 114 iv 3.4.3 R-MGMT preferentially interacts with MDM2 in vivo 116 3.4.4 R-MGMT protects p53 from deactivation by MDM2 118 3.4.5 Relationship between R-MGMT and p53/MDM2 regulatory loop in ML-1 121 3.4.6 Discussions 124 Chapter Conclusion 128 Chapter References 132 Chapter Appendix (Publications) 165 v List of Publications Oh,H.K., Teo,A.K.C., Swa,H.L.F., Zou,H., Tan,E.H.H., Chuang,L.S.H., Yeo,W.L., Choy,R.K.W., Fang,LL., Ali,R.B., Li,B.F (2004) MGMT/BRCA1 in transcription regulation of the Estrogen Receptor upon oxidative damage (Submitted) Chuang,L.S., Tan,E.H., Oh,H.K., and Li,B.F (2002) Selective depletion of human DNA-methyltransferase DNMT1 proteins by sulfonate-derived methylating agents Cancer Res 62, 1592-1597 Teo,A.K*., Oh,H.K*., Ali,R.B., and Li,B.F (2001) The modified human DNA repair enzyme O(6)-methylguanine-DNA methyltransferase is a negative regulator of estrogen receptor-mediated transcription upon alkylation DNA damage Mol Cell Biol 21, 7105-7114 ( *equal contributions) Ali,R.B., Teo,A.K., Oh,H.K., Chuang,L.S., Ayi,T.C., and Li,B.F (1998) Implication of localization of human DNA repair enzyme O6-methylguanineDNA methyltransferase at active transcription sites in transcription-repair coupling of the mutagenic O6-methylguanine lesion Mol Cell Biol 18, 16601669 Oh,H.K., Teo,A.K., Ali,R.B., Lim,A., Ayi,T.C., Yarosh,D.B., and Li,B.F (1996) Conformational change in human DNA repair enzyme O6-methylguanine-DNA methyltransferase upon alkylation of its active site by SN1 (indirect-acting) and SN2 (direct-acting) alkylating agents: breaking a "salt-link" Biochemistry 35, 12259-12266 Ayi,T.C., Oh,H.K., Lee,T.K., and Li,B.F (1994) A method for simultaneous identification of human active and active-site alkylated O6-methylguanine-DNA methyltransferase and its possible application for monitoring human exposure to alkylating carcinogens Cancer Res 54, 3726-3731 vi Abbreviations 6BG O6-Benzylguanine 6RG O6-alkylguanine ATCC American Type Culture Collection BCNU 1,3-bis(2-chloroethyl)-1-nitrosourea bp basepair BRCA1 Breast cancer susceptibility gene-1 Cdex Dextran-coated charcoal treated serum ChIP Chromatin immunoprecipitation Dex dexamethasone DMS dimethyl sulphate DMSO dimethyl sulphoxide DNA deoxyribonucleic acid DTT Dithiothreitol E.coli Escherichia coli E2 17-β-estradiol ECL enhanced chemiluminescence EDTA ethylene diamine tetra-acetic acid ERα Estrogen receptor-α ERE Estrogen receptor response element Fig figure FITC fluoresceine isothiocynate G gram GR Glucocorticoid receptor GRE Glucocorticoid receptor response element GSH gluthathione GST gluthathione-s-transferase H2O2 Hydrogen peroxide vii hr hour HRE Hormone response element IgG immunoglobulin g IP immunoprecipitation kDa kilodalton M molar Mab monoclonal antibody MeI Methyl iodide MGMT O6-methylguanine-DNA methyltransferase ml milliliter mM millimolar mRNA messenger ribonucleic acid NaCl sodium chloride NMU n-methylnitrosourea NP-40 nonindet-p-40 NR Nuclear receptor Pab polyclonal antibody PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction PMSF Phenylmethylsulphonyl fluoride R-MGMT Active-site alkylated MGMT SDS sodium dodecyl sulphate SV40 Simian virus 40 Tris Tris(hydroxymethyl)aminomethane TW-20 Tween 20 uM Micromolar viii Summary Alkylation of DNA at the O -position of guanine can lead to mutation and cell death These DNA adducts are repaired by the repair protein MGMT (O -methylguanine-DNAmethyltransferase) MGMT protein is conserved through evolution though the exact physiological function remains obscure MGMT acts by transferring the O -alkyl group to the cysteine residue at position 145 at its active site This repair process (indirect alkylation), as well as direct alkylation at its active site, irreversibly inactivates the protein, generates active-site alkylated MGMT, R-MGMT, and hence the term ‘suicidal’ repair R-MGMT adopts a conformation change which renders the protein susceptible to protease V8 cleavage In addition, the altered conformation exposes the domain surrounding the residues 91-107 containing the monoclonal antibody 3C7 recognition epitope We have thus far characterized the roles of R-MGMT in regulating the cell growth We have showed that R-MGMT, with its exposed domain, containing the LXXLL motif, interacts directly with the estrogen receptor alpha (ERα) to repress ERα− mediated transcription and proliferation in cells expressing both MGMT and ERα proteins We have also identified a role of active MGMT as a potential transcription activator of the ERα gene Chromatin Immunoprecipitation (CHIP) experiments demonstrated the association of MGMT with ERα gene in MCF7 breast cancer cell, and this interaction is disrupted and displaced by BRCA1 (Breast cancer susceptibility gene) protein when cells are exposed to oxidative damage These findings suggest that MGMT/R-MGMT play important roles in breast cancers Furthermore, it was showed ix Piette,J., Neel,H., and Marechal,V (1997) Mdm2: keeping p53 under control Oncogene 15, 1001-1010 Pike,A.C., Brzozowski,A.M., Hubbard,R.E., Bonn,T., 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Dev 12, 3488-3498 Zwijsen,R.M., Wientjens,E., Klompmaker,R., van der,S.J., Bernards,R., and Michalides,R.J (1997) CDK-independent activation of estrogen receptor by cyclin D1 Cell 88, 405-415 164 Oh et al -1- MGMT/BRCA1 in transcription regulation of the Estrogen Receptor upon oxidative damage Hue-Kian Oh*, Alvin K.C Teo*, Hannah L.F Swa, Hao Zou, Eileen H.H Tan, Linda, S.H Chuang, Wan-Lin Yeo, Richard K.W Choy, Lu-Lin Fang, Rahmen B Ali, Benjamin F L Li† H.K Oh, A.K.C Teo, H.L.F Swa, H Zou, E.H.H Tan, L.S.H Chuang, W.L Yeo, R.K.W Choy, L.L Fang, R B Ali, B.F.L Li, Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore B.F.L Li, Department of Physiology, Faculty of Medicine, National University of Singapore, Blk MD9, Medical Drive, Singapore 117597, Republic of Singapore †To whom correspondence should be addressed E-mail: mcblib@imcb.nus.edu.sg Phone: 65 68743763 and 65 68743797, Fax: 65 7791117 *H.K Oh and A.K.C Teo contribute equally to this work [CANCER RESEARCH 62, 1592–1597, March 15, 2002] Advances in Brief Selective Depletion of Human DNA-Methyltransferase DNMT1 Proteins by Sulfonate-derived Methylating Agents1 Linda Shyue-Huey Chuang, Eileen Hwee-Hong Tan, Hue-Kian Oh, and Benjamin F-L Li2 Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology [L S-H C., E H-H T., H-K O., B F-L L.], Department of Physiology, Faculty of Medicine [B FL L.], National University of Singapore, Singapore 117609, Republic of Singapore Abstract 5-Methylcytosine residues in the DNA (DNA methylation) are formed from the transfer of the methyl group from S-adenosylmethionine to the C-5 position of cytosine by the DNA-(cytosine-5) methyltransferases (DNMTs) Although regional hypermethylation and global hypomethylation of the genome are commonly observed in neoplastic cells, how these aberrant methylation patterns occur remains unestablished We report here that sulfonate-derived methylating agents, unlike N-methylnitrosourea or iodomethane, are potent in depleting DNMT1 proteins in human cells, in addition to their DNA-damaging properties Their effects on cellular DNMT1 are time and dosage dependent but independent of cell type Unlike ␥-irradiation, these agents apparently not activate the p53/p21WAF1 DNA damage response pathway to deplete the DNMT1 proteins because cells with wild-type, mutated, or inactivated p53 behave similarly However, cell cycle analysis and protease assay studies strongly suggest that methylmethanesulfonate may activate a cellular protease to degrade DNMT1 These results explain why reported observations on the effect of alkylating agents on DNMT1 activities in human cells vary significantly and provide a crucial link to understand the mechanism behind genomic hypomethylation Introduction Although the 5-MeC3 residues constitute only 2–3% of the mammalian genome, they serve important roles in gene regulation such as imprinting, long-term silencing of tissue-specific genes, and X-chromosome inactivation 5-MeC (DNA methylation)-mediated silencing may be accomplished by methylated cytosine binding proteins (i.e., MeCP2), which target histone deacetylase to condense the region of DNA containing 5-MeC (1), or the inability of transcription factors to bind methylated promoter sites (2, 3) Aberrant methylation, in the forms of regional hypermethylation (4) and global hypomethylation (5) of the genome, are commonly observed in neoplastic cells They are nonrandom and tumor type-specific (6), suggesting that improper methylation contributes to cancer initiation or progression Two causative roles for methylation in tumor progression have been observed: (a) silencing of tumor suppressor genes, such as p21WAF1, p16INK4a, or Rb, correlate with hypermethylation of their promoter regions in certain tumors (7); and (b) increased deletion mutation rates leading to genomic instability are associated with global hypomethylation of the genome (8) The observation that a DNMT gene is mutated in the ICF (immunodeficiency, centromeric instability, facial abnormalities) syndrome, which is characterized by hypomethylation and chromosome breakage (9 –12), strengthens the role of DNA methylation in human diseases How aberrant methylation occurs in tumor cells remains unestablished A likely candidate for study is the DNA-(cytosine-5) methyltransferase DNMT1, which constitutes Ͼ90% of methylation activities in the mammalian cells (13, 14) It processes the maintenance methylase activities necessary for normal cell growth, as well as unusual de novo methylase activities (15) Therefore, any alterations in the cellular level of DNMT1 protein may potentially lead to changes in DNA methylation patterns In this study, we addressed the question of whether mutagens can affect the DNMT1 protein by studying the levels of DNMT1 in cells exposed to ␥-irradiation and alkylating mutagens We report here that sulfonate-derived methylating agents, such as MMS and DMS, are potent in depleting DNMT1 proteins in the human cells, a novel addition to their abilities in producing alkylation lesions, such as apurinic sites and single- and double-strand breakage in the DNA (16) This hitherto unknown property of sulfonate-derived methylating agents suggests the multiplicity of these agents in tumor-causing actions Although DNMT1 and p21WAF1 proteins are shown to share an inverse relationship throughout the cell cycle and after exposure to ␥-irradiation, this relationship is not observed during MMS treatment, which induces DNMT1 depletion in cells with Wt, mutated, or inactivated p53 Results from cell cycle analysis and protease assay suggest that the effects of MMS may be direct or indirect, possibly via the modification of DNMT1 or activation of a cellular protease to degrade DNMT1 proteins These results may explain why reported observations on the effect of alkylating agents on DNMT1 activities in the human cells vary significantly (17) because MMS and DMS have a distinctive effect on the cellular DNMT1 proteins in a time- and dosage-dependent manner as compared with NMU and MeI Materials and Methods Treatment of Cells with Mutagens All cell lines were obtained from American Type Cell Culture and cultured as specified by American Type Cell Culture MMS, MeI, DMS, and NMU were purchased from Sigma Chemical Co.-Aldrich The cells were grown to 50% confluence before commencing drug treatment Stock solutions of all drugs were made in serum-free media and added to the cells for the indicated times ␥-Irradiation was done using a 60 Co source at room temperature Cell Lysis and Western Analysis Cell pellets were lysed in a modified RIPA buffer [50 mM Tris (pH 8.0), 0.3 M NaCl, 10 mM EDTA, 50 mM NaF, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, and 0.2 mM phenylmethReceived 6/20/01; accepted 1/10/02 ylsulfonyl fluoride] by sonication The lysates were then centrifuged at 14,000 The costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked advertisement in accordance with rpm for 20 at 4°C (Eppendorf), and the supernatant was recovered Protein 18 U.S.C Section 1734 solely to indicate this fact concentrations were determined by Lowry assay Cell lysates (100 ␮g) were Supported in part by the National Science and Technology Board of Singapore resolved on 6.9 and 12% SDS-PAGE After electrophoresis, the proteins were To whom requests for reprints should be addressed, at National University of transferred to polyvinylidene difluoride membranes (Bio-Rad) Western analSingapore, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore Phone: (65) 874 3763, (65) 874 3797; Fax: (65) 779 1117, (65) yses were performed as described (18) Monoclonal antibody Mab.2E10 for 775 9582; E-mail: mcblib@imcb.nus.edu.sg DNMT1 was described previously (18) Mab.D01 for p53 and Mab.PC10 for The abbreviations used are: 5-MeC, 5-methylcytosine; MMS, methylmethanesulfonPCNA detections were from Santa Cruz Biotechnology Mabs to actin (clone ate; DMS, dimethylsulfate; NMU, N-methyl-N-nitrosourea; MeI, iodomethane; Mab, monoclonal antibody; FC, flow cytometry; Wt, wild type C4) and p21WAF1 (clone 70) were from Boehringer Mannheim and Transduc1592 MOLECULAR AND CELLULAR BIOLOGY, Oct 2001, p 7105–7114 0270-7306/01/$04.00ϩ0 DOI: 10.1128/MCB.21.20.7105–7114.2001 Copyright © 2001, American Society for Microbiology All Rights Reserved Vol 21, No 20 The Modified Human DNA Repair Enzyme O6-Methylguanine-DNA Methyltransferase Is a Negative Regulator of Estrogen Receptor-Mediated Transcription upon Alkylation DNA Damage ALVIN K C TEO, HUE KIAN OH, RAHMEN B ALI, AND BENJAMIN F L LI* Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609, Republic of Singapore Received 22 March 2001/Returned for modification 25 April 2001/Accepted 16 July 2001 Cell proliferation requires precise control to prevent mutations from replication of (unrepaired) damaged DNA in cells exposed spontaneously to mutagens Here we show that the modified human DNA repair enzyme O6-methylguanine-DNA methyltransferase (R-MGMT), formed from the suicidal repair of the mutagenic O6-alkylguanine (6RG) lesions by MGMT in the cells exposed to alkylating carcinogens, functions in such control by preventing the estrogen receptor (ER) from transcription activation that mediates cell proliferation This function is in contrast to the phosphotriester repair domain of bacterial ADA protein, which acts merely as a transcription activator for its own synthesis upon repair of phosphotriester lesions First, MGMT, which is constitutively present at active transcription sites, coprecipitates with the transcription integrator CREBbinding protein CBP/p300 but not R-MGMT Second, R-MGMT, which adopts an altered conformation, utilizes its exposed VLWKLLKVV peptide domain (codons 98 to 106) to bind ER This binding blocks ER from association with the LXXLL motif of its coactivator, steroid receptor coactivator-1, and thus represses ER effectively from carrying out transcription that regulates cell growth Thus, through a change in conformation upon repair of the 6RG lesion, MGMT switches from a DNA repair factor to a transcription regulator (R-MGMT), enabling the cell to sense as well as respond to mutagens These results have implications in chemotherapy and provide insights into the mechanisms for linking transcription suppression with transcription-coupled DNA repair integrity of the DNA as they are arrested at the bulky DNA lesions inflicted by mutagens (33) while processing along the DNA to carry out their functions However, what could be an effective signaling factor for the DNA containing subtle DNA lesions that not arrest the polymerases? DNA repair enzymes are molecular sensors of damaged DNA in the cell, since they recognize and repair damaged DNA It would be a very effective survival strategy if the same DNA repair enzyme could also be a signaling molecule as well as a regulator for the presence of damaged DNA in the cells The Escherichia coli ADA protein is a unique example, exhibiting these properties in protecting the bacteria from the cytotoxic effects of the phosphotriester lesions in the DNA that are induced by alkylating agents Upon repairing the phosphotriester lesions by transferring the alkyl group from the phosphotriester lesion to the active site of the phosphotriester repair domain at its N terminus, the alkylated protein becomes a transcription activator for its own synthesis This increases the amount of the ADA protein in the bacterial cell for protection against further damage from alkylating agents (38, 39) Unfortunately, such an elegant DNA repair and response pathway appears to be limited to the prokaryotes, since homologs of the ADA protein are not found in the eukaryotes (44) Nevertheless, the O6-methylguanine-DNA methyltransferase (MGMT), which has an alkyl transfer repair mechanism similar to that of the E coli ADA protein, is present in all organisms It protects cells from the mutagenic and cytotoxic effects of alkylating carcinogens (10) by transferring the alkyl group of Exposure to environmental mutagens, such as UV irradiation and N-nitroso compounds, accounts for 80% of the human cancer incidence (30) The effectiveness of our cells’ attempts to repair the DNA lesions inflicted by mutagens on our DNA before DNA replication is fundamentally linked to manifestation of the disease through this etiological pathway The p53 protein is critical here for maintaining genomic integrity, since its induction upon DNA damage enables the cell to acquire sufficient time to repair the damaged DNA by halting cell cycle progression through its effector, the cell cycle-dependent kinase inhibitor p21WAFI (11, 15) However, p53 appears to be only a downstream effector of this DNA damage response pathway in the cell, since cellular factors, such as the hChk1 and hChk2 (human homologs of the yeast RAD53 and CDS1 proteins), are shown to stabilize p53 through phosphorylation upon exposure to mutagens (6, 14, 35) While much is known about cell regulation where external stimuli are transduced via the membrane receptors and kinase cascades to activate the nuclear DNA (8), knowledge of reciprocal pathways through which DNA, when it is damaged, signals cellular response through immediate factors remains circumstantial The high-fidelity property of DNA and RNA polymerases enables them to serve as important signaling factors for the * Corresponding author Mailing address: Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Dr., Singapore 117609, Republic of Singapore Phone: (65) 874 3763 or (65) 874 3797 Fax: (65) 779 1117 or (65) 775 9582 E-mail: mcblib@imcb.nus.edu.sg 7105 MOLECULAR AND CELLULAR BIOLOGY, Mar 1998, p 1660–1669 0270-7306/98/$04.00ϩ0 Copyright © 1998, American Society for Microbiology Vol 18, No Implication of Localization of Human DNA Repair Enzyme O -Methylguanine-DNA Methyltransferase at Active Transcription Sites in Transcription-Repair Coupling of the Mutagenic O6-Methylguanine Lesion RAHMEN B ALI, ALVIN K.-C TEO, HUE-KIAN OH, LINDA S.-H CHUANG, TECK-CHOON AYI, AND BENJAMIN F L LI* Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609, Republic of Singapore Received 22 May 1997/Returned for modification 27 June 1997/Accepted 27 October 1997 DNA lesions that halt RNA polymerase during transcription are preferentially repaired by the nucleotide excision repair pathway This transcription-coupled repair is initiated by the arrested RNA polymerase at the DNA lesion However, the mutagenic O6-methylguanine (6MG) lesion which is bypassed by RNA polymerase is also preferentially repaired at the transcriptionally active DNA We report here a plausible explanation for this observation: the human 6MG repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is present as speckles concentrated at active transcription sites (as revealed by polyclonal antibodies specific for its N and C termini) Upon treatment of cells with low dosages of N-methylnitrosourea, which produces 6MG lesions in the DNA, these speckles rapidly disappear, accompanied by the formation of active-site methylated MGMT (the repair product of 6MG by MGMT) The ability of MGMT to target itself to active transcription sites, thus providing an effective means of repairing 6MG lesions, possibly at transcriptionally active DNA, indicates its crucial role in human cancer and chemotherapy by alkylating agents The N-nitroso compounds are carcinogens to which we are all exposed because they are synthesized naturally in our gastrointestinal tract They are also cytotoxic, and some of them, notably bis-chloroethylnitrosourea, are used in cancer chemotherapy They owe their carcinogenic and toxic properties to their ability to alkylate the 6-oxygen of guanine in DNA Cells protect themselves from these compounds with a DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT), which removes the alkyl group from the O6-alkylguanine and transfers it to a cysteine residue in the active site of the transferase (a suicidal repair protein) (37) Interestingly, the mutagenic O6-alkylguanine lesions are also preferentially repaired at transcriptionally active DNA This was shown recently by direct measurements of the O6-ethylguanine residues formed and repaired in specific genes of rat cells treated with Nethylnitrosourea (48), which agree with the findings for Escherichia coli, by quantifying the frequency of GC-to-AT point mutations resulting from the miscoding behavior of the O6methylguanine (6MG) residues formed in the DNA of bacteria exposed to methylating agents (12, 42) However, unlike the arrested RNA polymerase-DNA lesion complex-mediated removal of bulky lesions in the transcribing DNA strand by NER, the O6-alkylguanine base in DNA does not arrest RNA polymerase This results in directing the misincorporation of uridine at this site (12) To address the question of how 6MG residues are preferentially repaired at transcriptionally active DNA by MGMT, we report here a detailed immunocytological study of the distribution of the repair protein within the cell nucleus Studies with polyclonal antibodies specific for the N and C termini of MGMT show that MGMT is strongly localized in small foci (speckles or embedded structures) in the nucleus Although this strong localization was not seen when monoclonal antibodies (MAbs) to inner regions of the transferase were used, the evidence is that this localization is a genuine phenomenon DNA in unwound (active) chromatin at sites of transcription or replication is vulnerable to damage induced by chemicals and irradiation (3, 7, 32, 34) Left unrepaired, these DNA lesions affect cell survival First, they either inhibit DNA polymerase (11) or are miscoded by the polymerase during DNA replication (1, 30, 31) Second, they can halt RNA polymerase during transcription of active genes (44) For example, to overcome the possible lethal blockage of transcription due to the arrested RNA polymerase at the thymine-thymine (T-T) photodimer (or bulky DNA lesions) formed in the transcribing DNA strand by irradiation, bacteria use the MFD protein (a transcription repair coupling [TRC] factor), which interacts with the arrested RNA polymerase at the lesion and recruits the uvrABC repair proteins (bacterial nucleotide excision repair [NER] proteins) for its repair (41, 42) In eukaryotes, similar preferential repair of bulky DNA lesions in the transcribing DNA strand by the NER pathway, i.e., TRC, has been reported However, the details of the mechanism appear to be much more complicated than the prokaryotic counterpart since coupling of eukaryotic DNA repair to transcription should involve several stages, such as nucleosome remodelling (e.g., the yeast RAD26 protein as a Swi2/Snf2-like ATPase [50]), assembling of the multicomponent preinitiation complex (e.g., the RAD25 helicase as a subunit of TFIIH [50]), and possibly others (e.g., the unestablished role of the human ERCC6 protein as a DNA-dependent ATPase [43]) Furthermore, TRC may be interrelated between different DNA repair pathways as mismatch repair-defective human cells may lack TRC of the T-T photodimer by NER (33) * Corresponding author Mailing address: Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Dr., Singapore 117609, Republic of Singapore Phone: (65) 874 3797 Fax: (65) 779 1117 E-mail: mcblib @mcbsgs1.incb.nus.edu.sg 1660 Biochemistry 1996, 35, 12259-12266 12259 Conformational Change in Human DNA Repair Enzyme O6-Methylguanine-DNA Methyltransferase upon Alkylation of Its Active Site by SN1 (Indirect-Acting) and SN2 (Direct-Acting) Alkylating Agents: Breaking a “Salt-Link”?† Hue-Kian Oh,‡ Alvin K.-C Teo,‡ Rahmen B Ali,‡ Allan Lim,‡ Teck-Choon Ayi,‡ D B Yarosh,§ and Benjamin F.-L Li*,‡ Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology, National UniVersity of Singapore, Kent Ridge, Singapore 0511, Republic of Singapore, and Applied Genetics Inc., 205 Buffalo AVenue, Freeport, New York 11520 ReceiVed February 14, 1996; ReVised Manuscript ReceiVed June 13, 1996X Human O6-methylguanine-DNA methyltransferase (MGMT) repairs DNA by transferring alkyl (R-) adducts from O6-alkylguanine (6RG) in DNA to its own cysteine residue at codon 145 (formation of R-MGMT) We show here that R-MGMT in cell extracts, which is sensitive to protease V8 cleavage at the glutamic acid residues at codons 30 (E30) and 172 (E172), can be specifically immunoprecipitated with an MGMT monoclonal antibody, Mab.3C7 This Mab recognizes an epitope of human MGMT including the lysine 107 (K107) which is within the most basic region that is highly conserved among mammalian MGMTs Surprisingly, the K107L mutant protein is repair-deficient and readily cleaved by protease V8 similar to R-MGMT We propose that R-MGMT adopted an altered conformation which exposed the Mab.3C7 epitope and rendered the protein sensitive to protease V8 attack This proposal could be explained by the disruption of a structural “salt-link” within the molecule based on the available structural and biochemical data The specific binding of Mab.3C7 to R-MGMT has been compared with the protease V8 method in the detection of R-MGMT in extracts of cells treated with low dosages of methyliodide (SN2) and O6-benzylguanine Their identical behaviors in producing protease V8 sensitive R-MGMT and Mab.3C7 immunoprecipitates suggest that probably methyl iodide (an ineffective agent in producing 6RG in DNA) can directly alkylate the active site of cellular MGMT similar to O6-benzylguanine The effectiveness of MeI in producing R-MGMT, i.e., inactivation of cellular MGMT, indicates that this agent can increase the effectiveness of environmental and endogenously produced alkylating carcinogens in producing the mutagenic O6-alkylguanine residues in DNA in ViVo ABSTRACT: Alkylation of the exocyclic oxygen of guanine in DNA, whether by environmental exposure or by cancer chemotherapeutic agents, is repaired by O6-methylguanine-DNA methyltransferase [MGMT, reviewed by Yarosh (1985) and Pegg (1990)], which transfers the alkyl group from O6alkylguanine residues in DNA to its own cysteine centered in the active site This active site alkylated protein, RMGMT, is inactive and is not regenerated to MGMT In humans the expression of MGMT is barely inducible, and therefore the repair capacity of a cell is determined by the amount of MGMT and its constitutive rate of synthesis MGMT rapidly repairs O6-methyl- and ethylguanine residues in DNA, although longer chain or bulkier derivatives are repaired with lesser efficiency It is generally believed that the observed loss of MGMT repair activities, or the formation of R-MGMT, in mammalian cells treated with alkylating agents are the result of MGMT repair of the O6† This project is supported by the National University of Singapore and the National Science and Technology Board of Singapore * Corresponding Author FAX: +65 779 1117 E-mail: mcblib@ leonis.nus.sg ‡ National University of Singapore § Applied Genetics Inc X Abstract published in AdVance ACS Abstracts, September 1, 1996 Abbreviations: MGMT, O6-methylguanine-DNA methyltransferase; R-MGMT, active-site alkylated O6-methylguanine-DNA methyltransferase; GSTMGMT, glutathione-S-transferase fusion protein of MGMT; Mab, mouse monoclonal antibodies; NMU, N-methylnitrosourea; MeI, methyliodide; 6MG, O6-methylguanine; 6RG, O6-alkylguanine; 6BG, O6-benzylguanine; ECL, enhanced chemiluminescence; wt, wild type S0006-2960(96)00363-7 CCC: $12.00 alkylguanine residues produced in the DNA by alkylating agents, i.e., by an indirect pathway However, the observation that O6-benzylguanine (6BG), which does not damage DNA, can effectively inactivate MGMT in human cells at micromolar concentrations (Dolan et al., 1990) suggests that cellular MGMT can be directly alkylated at its active site without DNA damage It is of particular interest to examine whether alkylating agents, apart from damaging DNA, can directly inactivate cellular MGMT as compared to the observation in Vitro (Brent, 1986) This can be studied by comparing the effectiveness of low dosages of SN1 and SN2 alkylating agents in producing R-MGMT (or a loss of MGMT activity) in cells, since SN2 alkylating agents are inefficient in producing O6-alkylguanine in DNA (Lawley, 1984) This study is also relevant to human cancer because SN2 agents such as methyl halides are potential human carcinogens (Foster et al., 1985) which are common in the environment (Harper, 1985; Gschwend et al., 1985) Such study, however, requires a sensitive method to distinguish inactive R-MGMT from active MGMT R-MGMT is stable in purified preparations and is not spontaneously degraded (Belanich et al., 1994; Pegg et al., 1991) The fate of R-MGMT in ViVo, however, varies among cells Immunoreactive R-MGMT is lost within the first few hours from HT29 colon adenocarcinoma cells (Belanich et al., 1994; Pegg et al., 1991) and human rhabdomyosarcoma xenografts grown in mice (Belanich et al., 1996), whereas © 1996 American Chemical Society Cancer Res 1994 Jul 15;54(14):3726-31 Related Articles, Links A method for simultaneous identification of human active and active-site alkylated O6-methylguanine-DNA methyltransferase and its possible application for monitoring human exposure to alkylating carcinogens Ayi TC, Oh HK, Lee TK, Li BF Chemical Carcinogenesis Laboratory, National University Hospital, Singapore Cells resist the major mutagenic effects of alkylating agents by the action of O6methylguanine-DNA methyltransferase (MGMT), which transfers the alkyl (R) group of O6-alkylguanine, produced in DNA by these chemicals, to a cysteine residue in its active site (formation of R-MGMT) We demonstrate that cellular RMGMT (which represents a record or memory within the cells exposed to these chemicals) can be assayed by its sensitivity toward proteolysis by protease V8 The possible use of this assay for monitoring exposure to alkylating carcinogens was investigated by using cultured cells and a preliminary study with the use of human blood from normal subjects and patients undergoing chemotherapy Cultured cell experiments show that R-MGMT is sufficiently stable for the monitoring purpose and its level bears a dose-response relationship to the concentrations of the alkylating agent used Interestingly, experiments with blood from patients undergoing chemotherapy show a gradual formation of R-MGMT in 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and an induced MGMT deficiency in cyclophosphamide-treated patients The use of this methodology, which allows for the possible quantification of active MGMT (cellular DNA repair capacity) and R-MGMT (recent exposure) simultaneously, in monitoring human exposure to alkylating carcinogens is discussed ... change in human DNA repair enzyme O6- methylguanine -DNA methyltransferase upon alkylation of its active site by SN1 (indirect-acting) and SN2 (direct-acting) alkylating agents: breaking a "salt-link"... bromodomain and the third zinc finger SRC interacts at a SID (SRC interacting domain for binding SRC) domain and PCAF interacts at the CH3 domain Interestingly, p300 was recently shown to contain a... binding CR2 (residues 301329) contains a zinc-finger domain overlapping the Retinoblastoma (Rb) tumour 35 suppressor protein-binding site CR3 (residues 444-483) encodes the RING-finger domain