The biological role of AidB has long been uncertain. Our data demonstrate that AidB prevents DNA damage by alkylating agents and counteracts the block to transcription that results upon exposure to alkylating agents, especially in genes that are transcribed from promoters containing UP elements. These effects are seen after treatment with MMS, MNNG and ENNG, three alkylating agents that produce different DNA lesions or damage spectra [24
]. The result that ENNG damage is also prevented is especially interesting since ENNG lesions are repaired not only the by adaptive response repair system, but also by nucleotide excision repair in E. coli
The result that AidB can prevent DNA damage seems inconsistent with the result that loss of AidB function by a complete deletion has little or no effect on MMS or MNNG sensitivity of the mutant strain ( and S3
). However, effects of an aidB
mutation on DNA damage and mutagenesis were seen at sublethal doses of alkylating agents (–). Since Ada dependent aidB
induction is relatively weak compared to that of other adaptive response genes [13
], it is possible that AidB protein levels are too low to provide adequate protection against lethal doses of alkylating agents. The primary function of AidB may be to protect DNA from the low levels of alkylators that are produced as by-products of stationary phase metabolism [26
], a possibility that is consistent with the observation that aidB
is induced and expressed at elevated levels in stationary phase [10
The result that the AidB protein specifically binds to DNA sequences that include the UP element [11
] (see also ), suggests that the lack of increased sensitivity to high levels of alkylating agents in the aidB
mutant () may also be due to the fact that AidB only protects a subset of the genome, leaving other genes, including essential ones, exposed to DNA damage. The aidB
mutant phenotype is consistent with targeted repair or damage prevention and is analogous to the effect seen in strains that lack the ability to carry out transcription-coupled repair (TCR) of UV damage, the only other gene specific repair or damage prevention system currently known. A TCR deficient mfd
mutant shows only a modest decrease in cellular resistance to UV, but a dramatic reduction in the rate at which repair of active genes occurs [32
]. Thus, the AidB prevention mechanism appears to be a cellular strategy to preferentially protect a subset of genes. In this case the genes include ones important for basic metabolic processes and key DNA repair genes. AidB is targeted towards genes whose promoters have upstream UP elements. This includes genes such as most of the ribosomal RNA genes and many tRNA genes as well as several key DNA repair genes required for recovery from alkylation damage such as recA, polA, sulA, recN
operon, and aidB
The presence of a functional aidB gene protects UP element genes from alkylation damage and results in more efficient transcription in the presence of alkylating agents. lacZ fused to the two UP element containing rrn and ompF promoters are transcribed 10- and 6- fold more efficiently in the presence of an alkylating agent than lacZ fused to an rrn promoter whose UP element has been deleted, or the leuA promoter, which has no UP element. Although it is possible that AidB has regulatory effects on these genes, a lower level of template damage should clearly contribute to the transcription efficiency.
Promoters lacking an UP element, and thus not efficiently bound by AidB protein still show a slightly higher level of transcription in wild type versus aidB mutants upon alkylation (2.2 and 2-fold enhancement for rrnBΔUP and Pleu, ). It is unclear if this aidB-dependent enhancement of transcription in the presence of an alkylating agent represents some direct protection by aidB, or is an indirect effect of the elevated levels of ribosomes, tRNAs and possibly other components of the translational machinery that are transcribed at a higher levels in the aidB+ strain under these conditions. The observation that the protection of lacZ fused to the rrn promoter lacking an UP element and the observation that plasmid DNA shows better protection in wild type than in an aidB mutant strain (), suggests that there may be some general protection resulting from the presence of aidB, especially when it is highly expressed, or induced for a long period of time as in these experiments. Under these conditions AidB may initially protect the genes preferentially targeted, followed by other parts of the genome if AidB protein accumulates to sufficiently high levels. The precise mechanism of action of AidB remains to be determined, though it is possible that it provides protection of DNA adjacent to its preferred binding site, either by simply inactivating alkylating agents and reducing the local concentration, or by polymerizing into multimers that extend from the initial binding site. In the latter case, it is likely to protect both by shielding the DNA and by inactivating alkylators.
However, the MNNG resistance resulting from expression of the DNA binding deficient aidB
mutant protein, AidB(Δ440–541)
indicates that the mutant lacking DNA binding activity still functions to prevent alkylation mutagenesis. This observation makes it unlikely that AidB functions by simply binding and coating the DNA, thus preventing access by alkylators. The ability of the DNA binding defective AidB protein to prevent mutagenesis suggests that AidB is not a DNA repair protein, since DNA repair would be inhibited by lack of DNA binding activity. Instead, AidB is more likely to function to prevent damage by detoxifying alkylating agents, which could reduce DNA alkylation even in the absence of DNA binding activity by reducing the intracellular concentration of active alkylators. A role for AidB in alkylating agent detoxification is also consistent with earlier work on AidB and analysis of the structural features of the protein [10
]. Determination of the precise mechanism by which AidB may inactivate alkylating agents requires further work to examine the chemistry of the hypothetical process.
It is unclear how widespread preferential damage prevention mechanisms such as AidB are, if other prokaryotes and eukaryotes have similar damage prevention proteins, or if the strategy of preferential DNA protection extends to mechanisms that prevent damage by other agents. In E. coli
gene is highly expressed in stationary phase and prevents oxidative DNA damage. Unlike AidB, however, this protein is produced at very high levels and appears to function as a genome wide protective protein. It is unclear if it may also have a preference for specific sequences when it is expressed at lower levels [37
In this manuscript we demonstrate that aidB binds preferentially to UP element containing genes and preferentially protects them from alkylation damage. Additionally, the reduction in transcription normally seen in the presence of alkylators is much greater in the aidB mutant than in wild type, indicating that transcription capacity is preserved when aidB is functional. We further demonstrate that aidB overexpression reduces mutagenesis and that this does not require the DNA binding domain, suggesting that it does not repair DNA, but prevents damage from occurring.