O⁶-Methylguanine-DNA methyltransferase
(MGMT, alkyltransferase, AGT)

What is MGMT ?

Genomic DNA is constantly under attacked by exogeneous and endogenous genotoxins, notably alkylating agents. Alkylation of DNA occurs spontaneously or is induced by exogenic factors. Two common sources of alkylation are compounds found in food and compounds used as chemotherapeutic drugs. If not repaired, alkylation of DNA gives rise to mutations or cell death. The most abundant environmental alkylating substance is Dimethylnitrosamine (DMNA), which is formed during food preparation. Nitrosonornikotin (NNN) and 4-(Methylnitrosoamino)-1-(3-piridyl)-1-butanon (NNK) are the dominant alkylating agents produced during smoking. Beside environmental substances, drugs used in chemotherapy also act via alkylation of the DNA, thus alkylating agents are among the most widely used chemotherapeutic drugs in the treatment of human cancer. These agents include procarbazine, dacarbazine, streptozotocin and temozolomide.

Alkylation of DNA can occur at various sites; however alkylation of the O⁶-position of guanine has the strongest mutagenic potential. Depending on the electrophilic character of the substances, alkylation of DNA can occur via two types of nucleophilic reactions, namely the S_N1 and S_N2 reaction. Substances that act via the S_N1 reaction have a high selectivity for alkylation at the O⁶ or N2-position of guanine and substances that act via S_N2 reaction, have a high selectivity for alkylation at the N7-position of guanine. Weak S_N1 agents are more specific for alkylation at the N2 position and hard S_N1 agents alkylate predominantly the O⁶-position of guanine. Treatment of cells with these compounds gives rise to N-alkylated and O-alkylated purines and pyrimidines as well as phosphotriesters. One of the most critical O-alkylated lesions is O⁶-methylguanine (O⁶MeG) that is formed, depending on the agent, at different levels but reaches its maximum at 8% of total alkylations.

 DNA methylation sites

The methyl group at the O⁶-position of guanine is removed by the DNA repair enzyme O⁶-methylguanine-DNA methyltransferase (MGMT) in a one step methyl transfer reaction. DNA-Alkyltransferases like the mammalian MGMT are enzymes that perform damage reversal in a one step reaction. During this reaction the methyl-group from O⁶-methylguanine and O⁴-thymine are transferred from the alkylated base within the genomic DNA onto an internal cystein residue within the active center of the alkyltransferase. This results in the restoration of guanine and thymine within the genomic DNA. Beside methyl-groups, also several other alkyl-groups are transferred onto the enzyme. The efficiency of the transfer reaction decreases with the size of these adducts. The alkyl group transfer leads to irreversible inactivation of the alkyltransferase and targets it for ubiquitination and proteasome-mediated degradation. Therefore alkyltransferases are suicid enzymes.This methyl transfer inactivates MGMT and the enzyme is degraded by the proteosome. This so-called suicide  reaction implies that the cells' capacity for repairing O⁶MeG lesions depend on the pre-existing MGMT levels in the cell or the rate at which the cell can re-synthesize MGMT. MGMT is expressed at different levels in deferent organs within the same individual. There is also differences in MGMT expression between individuals. Tumors also exhibit  different MGMT expression levels, not only depending on tumor type but also occurrences.

The so-called suicide  reaction

DNA-Alkyltransferases are ubiquitously distributed in prokaryotic and eukaryotic organism. Interestingly enough, in plants alkyltransferases have not yet been detected.  In E. coli, resistance to alkylating agents is mediated by the induction of four genes ada, alkA, alkB and aidB. The Ada protein, which represents the first cloned DNA-alkyltransferase, is responsible for the repair of O⁶MeG, O⁴-thymine and phosphotriester. It is a key player in the activation of the so-called adaptive response. In this response, pretreatment of bacteria with methylating agents renders cells resistant to the toxic and mutagenic activity of a second treatment with the same agent. Unlike other DNA-alkyltransferases, the Ada protein contains two active centers with reactive cytosine residues at position 69 and 321 respectively. Cystein 321 binds alkyl groups from O⁶-alkylguanine and O⁴-thymine, performing the repair reaction, whereas cystein 69 binds alkyl groups from alkylated phosphotriesters. The binding of alkyl-groups to cystein 69 leads to a confomational change of the Ada protein. Upon this alteration Ada is able to bind and activate the promotor of genes involved in the adaptive response, namely ada itself, the glycosylase alkA, the iron dependent dioxygenase alkB and aidB. A second alkyltransferase identified in E. coli is the Ogt ptrotein, which contains only one active center and is constitutively expressed.

The human O⁶-methylguanine-DNA methyltransferase (MGMT; also known as ATase, AGT) acts similar to the E.coli Ogt protein. The MGMT gene is located at chromosome band 10q26, and consists of one non-coding and four coding exons that encode a protein of 207 amino acids with a molecular weight of 24 kDa.

When O⁶MeG is not repaired by MGMT, it may lead to a mispair with Thymine during the DNA replication cell cycle phase. In the following DNA replication cycle, Thymine will pair with Adenine. This is the mechanism whereby O⁶MeG give rise to the distinctive  G - C to A - T point mutations. It is thought that these point mutations are the driving force behind the carcinogenic effects of alkylating agents.

The cytotoxic properties of O⁶MeG are exerted via the following mechanism. Un-repaired O⁶MeG DNA lesions enter the S-phase of the cell cycle and mispair with Thymine. These mispaires are then subject to mismatch repair. The MSH2 and MSH6 heterodimer MutSa binds the mispair and is required for toxicity. In the absence of MSH2 or MSH6 cells become resistant to O⁶MeG at the expense of a higher mutation frequency.