Identifying functional roles for alkB in the adaptive response of Escherichia coli to alkylation damage

In 1977 a novel, inducible and error free DNA repair system in Escherichia coli came to light. It protected E. coli against the mutagenic and cytotoxic effects of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS), and was termed the 'adaptive response of E. coli to alkylation damage'. This response consists of four inducible genes; ada, aidB, alkA and alkB. The ada gene product encodes an 06-methylguanine- DNA methyltransferase, and is also the positive regulator of the response. The alkA gene product encodes a 3-methyladenine- DNA glycosylase, and aidB shows homology to several mammalian acyl coenzyme A dehydrogenases. The alkB gene forms an operon with the ada gene, and encodes a 24 kDa protein. AlkB has been conserved from bacteria to humans indicating its importance, and although first described in 1983 its precise functional role still remains unknown. This Ph.D. project describes advancements made in determining functional roles for the enigmatic alkB gene product. AlkB mutants are known to be highly sensitive to MMS. We report here that AlkB is not involved in repair of DNA strand breaks in vivo, or in repair of the major toxic lesion 3-methyladenine known to block DNA replication. A major step forward using bacteriophage host cell reactivation analyses has shown that AlkB is defective in repair of damaged single-stranded DNA. A thousand- fold defect was seen compared to a two- fold defect with damaged double stranded DNA. This result was specific to SN2 alkylating agents, and its activity found to be independent of other known DNA repair pathways. AlkB mutants on exposure to MMS showed a low mutation frequency, and in vitro studies using various DNA substrates ruled out methyltransferase, glycosylase or any lesion transferral activities. Purified AlkB protein was shown to bind to DNA, with a greater affinity towards methylated single stranded DNA. Finally, we observed a greater sensitivity of E. coli alkB cells to MMS in an exponential phase of growth compared to cells in stationary phase. Thus we propose that AlkB is likely to act at single-stranded DNA regions within cells, such as at active sites of transcription and at replication forks