MGMT is O-6-methylguanine-DNA methyltransferase OR methylguanine-DNA methyltransferase , one of the house keeping genes which is present in all human beings and gets expressed during DNA damage(8).It encodes enzyme Methylated-DNA--protein-cysteine methyltransferase, a DNA repair enzyme belonging to MGMT family(1).. Methylation of promoter region of this gene has been the reason for the cancer in Pharynx, lungs and prostate (2-4).
1.2 BASIC DETAILS:
Fig 1. X-RAY STRUCTURE OF HUMAN O-6- ALKYLGUANINE-DNA ALKYLTRANSFERASE (PDB ID: 1QNT)
The enzyme consists of 176 amino acid in a single chain A.
Link to nucleotide Sequence:
Methylated-DNA--protein-cysteine methyltransferase is a DNA repair enzyme and helps in preventing mutations and cell death by removing the mutagenic and cytotoxic adducts formed by alkylation of guanines by the carcinogen O6-methylguanine (6).
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It can remove very complex alkyl group like ethyl-, propyl-, butyl-, benzyl- and 2-chloroethyl groups.
It gives tumor resistance to alkylating chemotherapies (5).
Act as a biomarker in detecting the cancer (5).
This gene is ubiquitous and the amount varies in different cells and in tumor cells. The gene does not contain a TATA box or CAT box in its promoter region but has GC rich regions mainly in regions 823 -936 (89%) and 979-1031 (90%). The promoter region is similar to that of the promoter regions of housekeeping genes, i.e. it is needed in all cell and is least influenced by environmental factors. The promoter region is found in the 1157b of the gene. The sequence CCGCCC is the gene promoter region in the 53 bp region (of what?) ,aids as the region for the binding of RNA polymerase II (11).There are two putative AP (what is AP) binding sites for the gene AP-1and AP-2, two glucocorticoid response elements for the promoter region of the gene and activates the transcription . In the presence of carcinogens and in tumor cells it(WHAT?) gets transactivated (12). Many factors like ionization and chemicals like dexamethasone, 2-chloroethyl-/V-nitrosourea and other mutagens, ionization radiation induced MGMT-CAT activation(13).There by formation of mRNA,which translate into the enzyme methylguanine-DNA methyltransferase mainly involved in DNA repair mechanism.
MGMT initiates a very simple human DNA repair pathway. Alkylation of DNA at O6-G (Guanine),O4-T (Thymine) and O2-T positions by carcinogen O6-MeG (O6-Methylguanine)(7) is the first step towards cancer formation . The alkyl group from O6-MeG goes and binds with the O (6 ) position of the guanine and forms O6-ethyl guanine which forms O6- meG :T mis match, leading to a G: C A: T transition that is associated with both mutagenesis and carcinogenesis. At this times the enzymes comes in action ,it removes the alkyl group from the O(6) position of the guanine and attaches covalently to the enzyme's cystene residue present in the active site (Cys145 in the human protein) in a stoichiometric reaction . Finally the protein become inactive, since the enzyme loses its vital properties after the reaction it is called as suicide reaction (8, 10).
Fig 1: binding of Alkyl to the guanine and formation of O6- meG: T
DNA (containing 6-O-methylguanine) = DNA (without 6-O methylguanin
+ Protein L-cysteine + protein S-methyl-L-cysteine(1)
Fig 2: Removal of Alkyl group by enzyme (9)
The major carcinogen from tobacco smoke nitrosamine 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone also called nicotine-derived nitrosamine ketone; NNK) was found to be responsible for the methylation caused in the promoter region of many tumor suppressor genes which in turns inactivates the gene leading to cancer formation (14).The carcinogen from smoke 4-(methylnitrosamino) -1-(3-pyridyl)-1- butanone found in tobacco smoke forms O6-guanine alkyl adducts and is removed by MGMT(2). (str)
Mechanism Of promoter Methylation by NNK
Methylation in promoter region does not cause any alteration in the gene but inactivates its normal function. There is very less knowledge about the cause for promoter methylation in MGMT (15-18).There is a strong positive association between MGMT methylation and tobacco smoking (20).
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DNA methyltransferase 1 (DNMT1) is an enzyme which causes methylation in DNA and the accumulation of this enzyme in the nucleus, binds to the CpG promoter region leading to hyper methylation and was found in smokers. NNK is one such carcinogen from the cigarette which can cause accumulation of that enzyme. This enzyme is phosphorylated by glycogen synthase kinase 3Î² (GSK3Î²) which takes up Î²-transducin repeat-containing protein (Î²TrCP) causing degradation of DNMT1via ubiquitin-proteosome system. NNK activates the AKT pathway which inhibits GSK3Î², which is involved in the phosphorylation of its substrate protein (here DNMT1), which in turn recruit Î²TrCP to carry out DNMT1 degradation. NNK also facilitates translocation of Î²TrCP with the help of shuttling protein contributing to further accumulation of the DNMT .This abnormal accumulation causes hypermethylation in the CpG promoter region(14). (needs elaborate explanation)
NNK induced promoter methylation
Interaction Mechanism of MGMT with other gene
MGMT methylation leads to the mutation in p53 in lung cancer. Many carcinogens like PAHs, nitrosamines, BPDE, NNK from cigarette smoke form DNA adducts with guanines in the CpG site. Among this NNK was the strongest carcinogen which forms ethyl adducts at the O6 position of guanine and pyridyloxobutyl adduct, which in normal case is repaired by MGMT.As the MGMT is methylated and inactive, it cannot repair the gene as in normal state and cause G-A mutation in lungs leading to tumor.(16)
P16 is a cell growth regulatory gene often seen methylated in lung cancers along with MGMT in smokers. MGMT protect the other gene buy removing the carcinogen bound to the guanine of the gene. The methylation occurring in this gene promoter region makes the gene inactive thereby preventing the repair of p16 gene leading to the formation of cancer.p16 methylation here is formed by NNK (18-19).
Alteration in Pharynx and prostate
In Pharynx and prostate cancer this gene was found to be methylated in the promoter region associated to smoking (3, 4).