Inducible repair of phosphotriesters in Escherichia coli.

Abstract

*Extracts from-Eswherichia coli cells induced for the adaptive response have been prepared that are capable of repairing 06-methylguanine, 04-methylthymine, and the phosphotriesters produced on the DNA backbone by alkylating-agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The phosphotriesters are repaired by a methyltransferase distinct from the one that demethylates 06-methylguanine. We propose that this increased capacity to repair phosphotriesters accounts for much of the increased resistance to MNNG toxicity seen in cultures induced for the adaptive response.Escherichia coli exposed to sublethal doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) soon become substantially more resistant to both the mutagenic and the cytotoxic effects of MNNG and related alkylating agents (1). This phenomenon is referred to as the adaptive response (2) and is effected by the induction of several proteins. Resistance to the-mutagenic effects of alkylation is mediated by the induction of a protein that rapidly removes one of the major alkylation products, 06-methylguanine (06-MeGua), from the DNA of the alkylated cells (3). This protein is unusual in that it repairs the alkylated guanine base by simply transferring the methyl group from the o6 position of guanine to one of the cysteines of the protein (4, 5). This process leads to inactivation of the enzyme, and any further repair of 06-MeGua requires synthesis of another whole new protein. Such a protein is called a methyltransferase (4).The mechanism-by-which cells develop resistance to the toxicity of MNNG is less well understood. This resistance develops only if the cells have a full complement of DNA polymerase I (6), implicating excision repair in the process in some way. Recently, it has been reported that a DNA glycosylase that is active on 3-methylpurines is induced as part of the adaptive response (7). This glycosylase is the product of the alkA gene (8) and cells mutant in this gene are unable to develop resistance to the toxic effects of methyl methanesulfonate (MMS) (8). These results led to the suggestion that 3-methylguanine might be a potent lethal lesion and its removal, in cells induced for the adaptive response, might be responsible-for the increased survival of these cultures after exposure to alkylating agents (7,8).An alternative explanation for why cells induced for the adaptive response become more resistant to MNNG toxicity is presented in this paper. We have found that cells apparently produce a second methyltransferase in responseto MNNG. This protein does not repair 06-methylguanine but removes methyl groups from the phosphate oxygens of the DNA backbone. We suggest that this dealkylation facilitates a variety of excision-repair reactions the composite effect of which is increased survival. MATERIALS AND METHODSBacterial Strains. The strains used were E. coli B strains F26 (His-Thy-) (2) and two derivatives of this strain-BS21, which is constitutive for the adaptive response (9), and BS21R, which is an Ada-revertant of BS21.Reag...

In Vivo Formation and Persistence of Modified Nucleosides Resulting from Alkylating Agents

“…The induced bacterial enzyme has been reported to dealkylate 04-MeT as well (21)(22)(23). This is not true for the rat liver enzyme (24 …”

Regulation and Expression of the Adaptive Response to Alkylating Agents

“…However, tlle intact Ada protein has the ability to abstract two, rather than one, methyl groups from DNA to internal cysteine residues. The second methyl group is not derived from 06-methylguanine, but from an apparently innocuous lesion, a methylphosphotriester (57,61,62). The reaction of an alkylating agent with either of the two oxygen atoms not involved in the phosphodiester bond can generate a pair of isomers, in the R and S configura tion.…”

Induction of the alkA gene of Escherichia coli in gram-negative bacteria

“…The 06-methylguanine-DNA methyltransferase activity of Ada irreversibly transfers a methyl group from 06-methylguanine in the DNA to the Cys-321 residue in the carboxyl-terminal domain of the same Ada protein (9,10,26). The methylphosphotriester-DNA methyltransferase activity also irreversibly transfers a methyl group from one of the diastereoisomers of methylphosphotriesters in the DNA to the Cys-69 residue in the amino-terminal domain of the Ada protein (18,19,44). These two methyltransferase reactions are terminal; once a methyl acceptor site of Ada protein is occupied, it does not appear to be demethylated (34).…”