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
). 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
). 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
) 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 do 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
). 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 O6
-alkylguanine (6RG) formed in the DNA by alkylating carcinogens (20
) to the cysteine residue at its active site (28
). This repair mechanism, however, depletes instantly the MGMT activity in the cell as MGMT is converted to the active-site alkylated and inactive MGMT, R-MGMT (19
). Even though the presence of unrepaired 6RG lesions in the cellular DNA is detrimental, producing point mutations upon DNA replication (1
) or mutated mRNA that can be instantly translated into an altered protein (42
) upon transcription (12
), the MGMT suicidal repair is preserved through evolution (28
). Could R-MGMT serve as a unique molecular memory of exposure to alkylating carcinogens (3
), similar to the alkylated ADA protein in bacteria, and therefore provide some important cellular functions?
Several observations suggested that human MGMT and R-MGMT could regulate estrogen receptor (ER)-dependent activities. First, the ligand-bound nuclear receptor activates cell proliferation (31
), and therefore, its activity must be controlled upon DNA damage. Second, active MGMT localizes at the active transcription sites of RNA polymerase II-dependent genes (2
) (including ER-regulated genes). Third, biochemical analyses show that human R-MGMT adopts an altered conformation in exposing the VLWKLLKVV domain (26
) containing an LXXLL motif that also is found in transcription coactivators for their binding to nuclear receptors (13
). Fourth, the LXXLL motifs of R-MGMT and the coactivator glucocorticoid receptor interacting protein binding to the ligand binding domain of ER-α adopt similar amphipathic α-helix structures (7
). Finally, this LXXLL motif of human and mammalian MGMTs is not found in lower organisms that do not have ER.
Here we provide the experimental findings of how human R-MGMT serves as a DNA damage-induced transcription suppressor for regulating ER-mediated cell proliferation upon exposure to alkylating agents. These findings serve as an important example of how a DNA repair enzyme interplays with transcription factors and integrators to regulate cell proliferation upon DNA damage, and they provide a good reason for the suicidal repair of the 6RG lesions by MGMT to form R-MGMT. This appears to be a highly specific protein modification in the cell that enables the DNA to regulate itself upon alkylation damage by transducing the signal from the 6RG lesions in the damaged DNA via R-MGMT to block ER from activating the DNA directly to transcribe or indirectly to replicate by ER-transcribed gene products.