Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O ⁶ -alkylguanine adducts in Escherichia coli

Abstract

O 6 -alkylG adducts are highly mutagenic due to their capacity to efficiently form O 6 -alkylG:T mispairs during replication, thus triggering G→A transitions. Mutagenesis is largely prevented by repair strategies such as reversal by alkyltransferases or excision by nucleotide excision repair (NER). Moreover, methyl-directed mismatch repair (MMR) is known to trigger sensitivity to methylating agents via a mechanism that involves recognition by MutS of the O 6 -mG:T replication intermediates. We wanted to investigate the mechanism by which MMR controls the genotoxicity of environmentally relevant O 6 -alkylG adducts formed by ethylene oxide and propylene oxide. Recently, the alkyltransferase-like gene ybaZ (eATL) was shown to enhance repair of these slightly larger O 6 -alkylG adducts by NER. We analyzed the toxicity and mutagenesis induced by these O 6 -alkylG adducts using single-adducted plasmid probes. We show that the eATL gene product prevents MMR-mediated attack of the O 6 -alkylG:T replication intermediate for the larger alkyl groups but not for methyl. In vivo data are compatible with the occurrence of repeated cycles of MMR attack of the O 6 -alkylG:T intermediate. In addition, in vitro, the eATL protein efficiently prevents binding of MutS to the O 6 -alkylG:T mispairs formed by the larger alkyl groups but not by methyl. In conclusion, eATL not only enhances the efficiency of repair of these larger adducts by NER, it also shields these adducts from MMR-mediated toxicity.futile mismatch repair cycling | interference between repair pathways | nucleotide excision repair | ybaZ gene

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

“…The eATL cassette was isolated by PCR using the vector pMBP-YbaZ [22] as template and eATL forward: 5 -GCCATGCGACTTCACTCGGGC-3 ; reverse: 5 -TCAGTAGTTCCAGCGATAACG-3 primers. The version of eATL pcDNA3.1 containing a hygromycin resistance cassette was obtained by excision of the eATL sequence with Hind III and Xho I from the eATL pcDNA3.1 (neomycin) vector and insertion into the Hind III-Xho I digested pcDNA3.1 (hygromycin) vector.…”

“…In Schizosaccharomyces pombe, the alkyltransferase-like protein 1 (Atl1) targets O 6 MeG for global genome NER, whereas transcription-coupled NER participates in the repair of more complex adducts [20]. The ATL of Escherichia coli, initially described as ybaZ [21], was not only reported to initiate NER [17], but also to mask DNA damage and thus prevents the conversion of certain O 6 -alkylguanines to toxic lesions by the MMR system [22]. This process was shown to reduce the transforming effect in E. coli of a plasmid containing O 6 -hydroxyethyl-, O 6 -1-hydroxypropyl-and O 6 -2-hydroxypropylguanine, but not O 6 MeG, albeit at the cost of a higher mutation frequency [22].…”

“…The ATL of Escherichia coli, initially described as ybaZ [21], was not only reported to initiate NER [17], but also to mask DNA damage and thus prevents the conversion of certain O 6 -alkylguanines to toxic lesions by the MMR system [22]. This process was shown to reduce the transforming effect in E. coli of a plasmid containing O 6 -hydroxyethyl-, O 6 -1-hydroxypropyl-and O 6 -2-hydroxypropylguanine, but not O 6 MeG, albeit at the cost of a higher mutation frequency [22]. It was therefore proposed to be a lethality avoidance mechanism by damage tolerance.…”

The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

“…Other glycosylases that remove oxidized bases include AlkA (7,46), MUG (37,59), KsgA (71), and endonucleases III (55,62) and VIII, encoded by the nth gene (33) and the nei gene (36,48), respectively. Tag, Ogt, and the recently characterized YbzA remove alkylated bases (47), but it is possible that they may also contribute to the removal of as yet unidentified adducts. Among the oxygenase-or glycosylase-deficient strains, mutants deficient in AlkB display the greatest sensitivity to KAN, perhaps pointing to the ethenoadenine and ethanoadenine adducts resulting from hydroxyl radical-generated lipid peroxidation (13,25) as playing a key role in KAN-mediated cell death.…”

Alkyltransferase-like protein (Atl1) distinguishes alkylated guanines for DNA repair using cation–π interactions

“…3, 11, and 12) are highly homologous to AGTs but have a different amino acid (typically Trp or Ala) in place of the nucleophilic active site Cys. ATL proteins retain the ability to bind single-stranded or duplex DNA containing O 6 -alkylguanine, and although unable to undertake the de-alkylative repair reaction, they protect against the adverse effects of DNA alkylation damage by downstream recruitment of nucleotide excision repair (NER) (13)(14)(15)(16)(17)(18)(19)(20)(21)(22).…”