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

Abstract

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...

Mice over-expressing human O6alkylguanine-DNA alkyltransferase selectively reduce O6methylguanine mediated carcinogenic mutations to threshold levels after N-methyl-N-nitrosourea

“…Since, others have found a similar mutation spectrum after methylating agent exposure in actively transcribed genes such as hprt with preferential O 6 mG adduct repair on the transcribed strand (Lukash et al, 1991;Dobo et al, 1998) to that which we observed, and plasmid shuttle vector mutation analysis has also yielded similar mutation type changes to those observed here in the presence and absence of AGT (Moriwaki et al, 1991), we conclude that our ®ndings are representative of the impact of AGT on mutation frequency after MNU. This is further supported by the recent ®nding that nuclear localized AGT was present in transcriptionally active regions (Ali et al, 1998), perhaps coupled to nucleotide excision repair (Sitaram et al, 1997). While it is likely that the overall repair of O 6 mG adducts is lower in the inactive lacI gene than in actively transcribed genes, the mutation spectrum and type speci®c frequency di erences between the Big Blue TM and MGMT+/Big Blue TM mice are also likely to be predictive of the changes found in actively transcribed genes.…”

O6‐alkylguanine‐DNA alkyltransferase: Role in carcinogenesis and chemotherapy

“…Although evidence has been presented for the co-localisation of ATase with nascent RNA, (14) the rate of removal of O Phosphorylation of the protein, resulting in a decrease in activity, has recently been reported. (16) One exception to the general alkyl transfer repair mechanism is the action of the protein on N1-O 6 -ethenoguanine.…”

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

“…First, MGMT as a DNA repair factor is constitutively present at active transcription sites, whereas R-MGMT, as a transcription suppressor (Fig. 6E), appears instantly upon exposure to alkylating agents (2). Second, active MGMT is a component of the CBP/p300-containing complex (Fig.…”

“…4F) that integrates external signals to activate the transcription activities of nuclear receptors (40,43). Furthermore, CBP/p300 is a histone acetylase (17), which can modify the histone octamer in the nucleosome to poise the DNA for transcription (41) and also expose the DNA to undesirable damage by mutagens (2). Thus, it is strategic for MGMT to be a component of the CBP/p300-containing complex (Fig.…”