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 O 6 -methylguanine-DNA methyltransferase (R-MGMT), formed from the suicidal repair of the mutagenic O 6 -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.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 p21 WAFI (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 integrity of the DNA as they are arrested at the bulky DNA lesions inflicted by mutagens (33) whil...

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 that 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.

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 O 6 -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 O 6 -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.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).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, ar...