The Ada protein acts as both a positive and a negative modulator of Escherichia coli's response to methylating agents.

Abstract

The adaptive response of Escherichia coli protects the cells against the toxic and mutagenic effects of ceilain alkylating agents. The major effector molecule regulating this response is the 39-kDa Ada protein, which functions as both a DNA repair protein and a transcriptional activator. Ada removes methyl groups from phosphotriester and 0P-methyluanine lesions in DNA, irreversibly transferring them to cystehie residues at positions 69 and 321, respectively. When methylated at Cys-69, Ada is converted into a potent activator ofada and alkA transcription and binds to a sequence (Ada box) present in both promoters. We have found that physiologically relevant higher concentrations of unmethylated Ada are able to inhibit the activation of ada transcription by methylated Ada, both in vitro and in vivo. In contrast, the same concentrations of unmethylated Ada do not inhibit the activation of aLkA transcription by methylated Ada, either in vitro or in vivo.Deletion of the carboxyl-trminal 67 amino acids of Ada abolished the ability of the unmethylated form of the protein to inhibit activation of ada transcription but not the ability of the methylated form to activate ada or alkA transcription. Our results suggest that the Ada protein plays a pivotal role in the negative modulation of Its own synthesis and therefore in the down-regulation of the adaptive response. Elements present in the carboxyl terminus of Ada appear to be necessary for this negative regulatory function.The adaptive response of Escherichia coli is an inducible DNA repair system that helps prevent the mutagenic and cytotoxic effects caused by exposure to alkylating agents, particularly methylating agents (1-3). The Ada protein removes methyl groups from the premutagenic lesions 06-methylguanine and 04-methylthymine, transferring them irreversibly to Cys-321, a cysteine located in its carboxylterminal domain (4-6), and also removes methyl groups from the S diastereoisomers of DNA methylphosphotriesters, transferring them irreversibly to Cys-69, which is located in its amino-terminal domain (7-10). Methylation at Cys-69 converts Ada into a strong transcriptional activator ofits own gene (ada) and also of several other genes (alkB, alkA, and aidW) (11)(12)(13)(14)(15) N-methyl-N'-nitro-N-nitrosoguanidine. 9730The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Alteration of lysine 178 in the hinge region of the Escherichia coli ada protein interferes with activation of ada, but not alkA, transcription

“…We have previously demonstrated that physiologically relevant concentrations of unmethylated Ada protein are able to inhibit methylated Ada-promoted ada transcription both in vitro and in vivo (18). This ada-specific inhibition does not occur at the alkA promoter either in vitro or in vivo.…”

“…␤-Galactosidase assays were performed as described elsewhere (18). Cells were grown in M9 minimal medium (12) to an optical density (OD) of 0.2 to 0.3.…”

“…Purification of wild-type Ada, Ada-178 (containing the first 178 amino acids) and AdaK178P (containing a lysine-to-proline mutation at position 178) were purified exactly as described earlier (18). Methyltransferase assays were performed as described previously (1).…”

“…Methylated Micrococcus luteus DNA substrate was prepared by the procedure described by Nakabeppu and Sekiguchi (17). In vitro transcription experiments, production of template DNAs, and methylation of proteins were performed exactly as previously described (18).…”

“…We have recently reported in vitro and in vivo evidence suggesting that once all the repairable methylphosphotriesters have been repaired via methyl transfer to Ada, the unmethylated Ada molecules that accumulate, because of the autoregulatory circuit, play a key role in shutting off the adaptive response (18). We have shown that this negative modulatory role of unmethylated Ada protein occurs specifically at the ada promoter and not at alkA.…”