The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity

Abstract

In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O(6)-methylguanine (O(6)MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O(6)-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown. Here we expressed the ATL protein of Escherichia coli (eATL) in mammalian cells and addressed the question whether it is able to protect them against the cytotoxic effects of alkylating agents. The Chinese hamster cell line CHO-9, the nucleotide excision repair (NER) deficient derivative 43-3B and the DNA mismatch repair (MMR) impaired derivative Tk22-C1 were transfected with eATL cloned in an expression plasmid and the sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was determined in reproductive survival, DNA double-strand break (DSB) and apoptosis assays. The results indicate that eATL expression is tolerated in mammalian cells and conferes protection against killing by MNNG in both wild-type and 43-3B cells, but not in the MMR-impaired cell line. The protection effect was dependent on the expression level of eATL and was completely ablated in cells co-expressing the human O(6)-methylguanine-DNA methyltransferase (MGMT). eATL did not protect against cytotoxicity induced by the chloroethylating agent lomustine, suggesting that O(6)-chloroethylguanine adducts are not target of eATL. To investigate the mechanism of protection, we determined O(6)MeG levels in DNA after MNNG treatment and found that eATL did not cause removal of the adduct. However, eATL expression resulted in a significantly lower level of DSBs in MNNG-treated cells, and this was concomitant with attenuation of G2 blockage and a lower level of apoptosis. The results suggest that eATL confers protection against methylating agents by masking O(6)MeG/thymine mispaired adducts, preventing them from becoming a substrate for mismatch repair-mediated DSB formation and cell death.

Alkyltransferase-like protein clusters scan DNA rapidly over long distances and recruit NER to alkyl-DNA lesions

“…However, protection was conveyed predominantly via shielding of alkyl-guanine-induced alkylG:T mispairs from futile rounds of DNA mismatch repair that result in DNA double strand break formation, as also shown in E. coli (36,37). ATL did not initiate NER of methyl-guanine in mammalian cells in these studies (36). Consistently, in E. coli enhanced NER by ATL could only be observed for larger alkyl lesions and not for methyl-guanine (37,38).…”

“…Finally, previous studies showed that expression of E. coli ATL protects mammalian cells against cytotoxicity of methyl-guanine lesions (36). However, protection was conveyed predominantly via shielding of alkyl-guanine-induced alkylG:T mispairs from futile rounds of DNA mismatch repair that result in DNA double strand break formation, as also shown in E. coli (36,37).…”

Redox regulation of genome stability by effects on gene expression, epigenetic pathways and DNA damage/repair

“…Reactive oxygen and nitrogen species predominantly induce specific base modification, such as 8-oxo-dG, 8-nitro-dG [9] , or GC to TA transversions due to their high reactivity with strong nucleophilic sites on nucleobases [10] . External chemical compounds can introduce particular chemical groups, for example alkylation of DNA by methylnitrosourea [11] or N-methyl-N′-nitro-N-nitrosoguanidine [12] can cause DNA cross-links (e.g. mitomycin C, cisplatin) [13,14] , or enhance formation of single- and double-strand breaks by sealing DNA-topoisomerase complexes [15] .…”

A Chemical Link between Meat Consumption and Colorectal Cancer Development?

“…Repair of O 6 -CMG was first investigated by assessing azaserine mutagenicity in bacteria and yeast with defined deficiencies in repair factors. , Alkyltransferase repair enzymes and NER assisted by alkyltransferase-like (ATL) enzymes ,, protected bacterial and yeast cells from mutations resulting from exposure to azaserine. ATL proteins are homologous to O 6 -methylguanine-DNA methyltransferase (MGMT), but do not have the transferase domain; therefore, they bind DNA at modified bases but do not catalyze their removal. − Binding and conformational studies supported that ATL proteins serve as signaling factors of O 6 -alkylG, including O 6 -CMG, to the NER pathway. ,, Although ATLs have not been found in higher eukaryotes yet, these results raise the question of whether alkyltransferases such as MGMT might have a similar role in facilitating and signaling for the repair of O 6 -CMG in higher organisms. However, this speculation has never been confirmed, and the role of MGMT in relation to O 6 -CMG persistence in human cells remains equivocal.…”