Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O6-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.

Fig. 1.

Experimental outline. (A) O⁶-alkylguanine adducts used in the present study: O⁶-methylguanine (mG), O⁶-hydroxyethylguanine (heG), O⁶-1-hydroxypropylguanine (1hpG), and O⁶-2-hydroxypropylguanine (2hpG). (B) The relative transformation efficiency characterizes the toxicity of a single adduct during replication. For this purpose, single-stranded plasmid constructs are transformed into E. coli cells. Transformants are selected on ampicillin-containing plates. The relative transformation efficiency is determined as the ratio of colonies produced by a given amount of adduct-carrying plasmid DNA over the same amount of control plasmid DNA. (C) Determination of the induced mutant fraction. A 14-mer oligonucleotide (dotted rectangle) that carries the O⁶-alkylguanine adduct on the third G within an SmaI restriction site (underlined) was inserted by ligation into a gapped-duplex structure as described in Materials and Methods. Following introduction into E. coli, the transformation mixture is cultivated in ampicillin-containing LB medium and the plasmid pool is extracted. During replication, the O⁶-alkylguanine adduct will mostly mispair with T and yield SmaI-resistant plasmid progeny (SmaI^R). In contrast, accurate repair before replication or occasional C insertion during replication will yield SmaI-sensitive plasmid progeny (SmaI^S). In addition, with double-stranded constructs, replication of the undamaged strand yields SmaI^S plasmid progeny. The mutant fraction can be quantified following agarose gel electrophoresis as the ratio of the intensities SmaI^R/(SmaI^S + SmaI^R).

Fig. 2.

eATL protects O⁶-alkylguanine:T mispairs from MMR-mediated toxic processing. Single-stranded plasmid vectors carrying a single O⁶-alkylguanine adduct were introduced into various E. coli strains by transformation. All strains used are defective in both AT (ada, ogt) and NER (uvrA) repair and additionally carry a mutation in mutS (MMR⁻) or ybaZ (ATL⁻). (A) The RTE is determined as the efficiency of the single-stranded plasmid carrying a given adduct to form colonies on ampicillin plates over the efficiency of the same quantity of lesion-free control construct. In an MMR⁻ strain, all adducts exhibit an RTE of ≈80%, illustrating the low replication-hindering capacity of these adducts. In an MMR-proficient strain, survival of the mG adduct is strongly reduced (<10%), whereas it remains high for all other adducts (50–60%). Interestingly, inactivation of ybaZ (ATL⁻) strain strongly sensitizes all adducts to levels < 10%. It can thus be concluded that eATL efficiently protects all adducts (except mG) from the attack by MMR of the O⁶-alkylguanine:T replication intermediate. (B) Determination of the mutant fraction induced by the O⁶-alkylguanine adducts as described in Materials and Methods. In an MMR-defective background, all adducts exhibit mutant fractions close to 100%. When MMR processing is proficient (AT⁻NER⁻ATL⁺MMR⁺), the mutant fraction is strongly decreased for mG but not for the other adducts. Further inactivation of eATL sensitizes all adducts to MMR (AT⁻NER⁻ATL⁻MMR⁺).

Fig. 3.

Modulation by eATL and MMR of the induced mutant fraction using double-stranded DNA probes. The elevated mutant fraction (30–35%) induced by all adducts in the MMR-defective strain is severely reduced when MMR is proficient. This reduction is less pronounced for heG and hpG adducts compared with mG except when eATL is also inactivated. Processing by MMR of the mutagenic G*:T replication intermediates is thus counteracted by eATL for heG and hpG but not mG, reflecting the differential affinity of eATL for the various adducts in vitro.

Fig. 4.

eATL prevents binding of MutS to O⁶-alkylguanine:T mispairs. The incubation buffer for MutS binding assays contains cold competitor DNA (1 kb ladder; New England Biolabs) at a concentration of 10 ng/mL to minimize unspecific binding of MutS to double-stranded DNA. The DNA probes are ³²P-radiolabeled oligoduplexes containing G, mG, heG, 1hpG, and 2hpG, paired to C (:C) or to T (:T). Oligonucleotides were first incubated with eATL at 0 nM (lanes 2 and 8), 125 nM (lanes 3 and 9), 250 nM (lanes 4 and 10), and 500 nM (lanes 5, 6, 11, and 12). Reactions were next supplemented with MutS at a final concentration of 1 μM (lanes 2–5 and 8–11). eATL interferes with MutS binding for all adducts except mG. The same results are observed when the order of addition of eATL and MutS is reversed.

Fig. 5.

How eATL interferes with MMR-mediated processing of O⁶-alkylG:T replication intermediates. eATL binds to O⁶-alkylguanine-containing oligonucleotides and thus inhibits MutS protein binding to the G*:T mispair. As a consequence, eATL prevents MMR-mediated processing of the G*:T replication intermediates. Data presented in the present paper show that MMR-mediated toxicity involves MutH functions rather than just MutS binding and suggest that repeated cycles of processing trigger toxicity in E. coli. The effect of eATL is observed for the larger O⁶-alkylG adducts (hydroxyethyl and hydroxypropyl) but not for methyl, in good agreement with the respective binding affinities of eATL for the different adducts.