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”?

Abstract

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.

Implication of Localization of Human DNA Repair Enzyme O6-Methylguanine-DNA Methyltransferase at Active Transcription Sites in Transcription-Repair Coupling of the Mutagenic O6-Methylguanine Lesion

“…These results suggested that, upon the repair of 6MG, MGMT undergoes a conformation change in vivo as indicated by the masking of these previously exposed N and C termini. This agrees with the specific cleavage of R-MGMT by protease V8 in vitro at glutamic acid residues E30 and E172 flanking the N and C termini of MGMT (35).…”

“…To understand whether the rapid disappearance of MGMT speckles upon NMU treatment was due to DNA damage and repair by MGMT, we analyzed the amount of R-MGMT formed in these NMU-treated cells and the ability of MGMT.PAb to immunoprecipitate active MGMT and R-MGMT. The R-MGMT was quantified by the protease V8-Western blot method (35), by which R-MGMT appeared as two distinctive 14-and 18-kDa polypeptides after cleavage by protease V8. Figure 6c, subpanel A, shows that the levels of total cellular MGMT remain constant (therefore, no degradation) in all samples.…”

The Modified Human DNA Repair EnzymeO6-Methylguanine-DNA Methyltransferase Is a Negative Regulator of Estrogen Receptor-Mediated Transcription upon Alkylation DNA Damage

“…GST-(wt) and MGMT (K107L) were prepared as described previously (26). Recombinant ER-␣ (50 ng; Oncogene Science) was added to buffer B (100 l of 50 mM HEPES [pH 7.3], 0.1% Triton X, 10% glycerol, 100 mM KCl, and 1 mM dithiothreitol) with glutathione-Sepharose (20 l of a 50% suspension)-bound fusion proteins (ϳ100 ng) which were first treated with bovine serum albumin (100 l of 20% bovine serum albumin in buffer B) at 4°C for 1 h. After shaking at 4°C for 15 min, the beads were recovered and washed three times with 500 l of buffer B before boiling in Laemmli buffer for immunoblot analysis.…”

“…To investigate human R-MGMT function, we treated some MGMT-positive (mer ϩ ) and -deficient (mer Ϫ ) cells with 6BG, which alkylates selectively the cysteine C145 residue of human MGMT without causing DNA damage (26). Flow cytometry (FC) analysis of these 6BG-treated cells in Fig.…”

“…2C and F). Thus, modification of MGMT may be causal in blocking the activation of ER by E2, because 6BG and MeI convert MGMT to R-MGMT via independent pathways (26).…”